Embryonic cell populations and methods to isolate such populations

ABSTRACT

The present invention relates to novel immortalized precursor cell populations derived from embryonic stem cell populations and methods to produce such cell populations. Also disclosed is an assay to identify regulatory compounds capable of controlling cell growth for therapeutic and experimental use.

GOVERNMENT RIGHTS

This invention was made in part with government support under HL48834-02, awarded by the National Institutes of Health. The governmenthas certain rights to this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/343,686 for "Novel Embryonic Cell Populationsand Methods to Isolate Such Populations", filed Nov. 21, 1994,incorporated herein by this reference in its entirety. The presentapplication is also a continuation-in-part of PCT patent applicationSer. No. US95/14495 for "Novel Embryonic Cell Populations and Methods toIsolate Such Populations", filed Nov. 20, 1995, incorporated herein bythis reference in its entirety.

FIELD OF THE INVENTION

The present invention is related to novel populations of precursor cellsand methods to produce such populations of cells. More particularly, thepresent invention relates to a population of immortalized precursorcells and the use of such populations to identify compounds capable ofregulating the growth of a cell.

BACKGROUND OF THE INVENTION

Multicellular animals are derived from a clone of cells descended from asingle original cell, the fertilized egg. Embryogenesis involves thedivision and differentiation of multipotential cells, each cell havingthe ability to develop into multiple cellular lineages. Phenotypically,the cells of such lineages can vary substantially, such as blood cells,muscle cells and neural cells, being specialized.

A wide spectrum of diseases may be treated based upon both thepossession of a population of cells having multi-lineage potential andan understanding of the mechanisms that regulate embryonic celldevelopment. For example, the capacity to generate a new population ofhematopoietic cells is the basis of bone marrow transplantation, whichis currently used as a treatment for a growing number of diseasesincluding anemia, leukemia and breast cancer. In addition,transplantation of genetically altered multipotential cells has beenconsidered as potential therapy for a variety of different diseasesincluding AIDS.

One of the major barriers to both the treatment of diseases and thestudy of the process by which an undifferentiated embryonic cell becomescommitted to a particular developmental pathway is the lack of access topopulations of cells that are sufficiently multipotent to be able todevelop into various lineages. In particular, much attention has beenpaid to the use of bone marrow stem cells as a source of multi-potentialcells for therapy and experimental use. Bone marrow stem cells, however,have limited use because such populations of cells comprise asubpopulation of complex hematopoietic tissue and, therefore are rare.In addition, bone marrow stem cells have not been grown as asubstantially homogeneous population in tissue culture.

Following fertilization, an egg divides over a period of days to form ablastocyst. A blastocyst includes a hollow ball of cells having an innercell mass and a fluid-filled cavity, both encapsulated by a layer oftrophoblast cells. The blastocyst then implants into the uterine walland enters into the embryonic stage of development characterized by theformation of the placenta, the development of major internal organs andthe appearance of major external body structure.

Cells from the inner cell mass of an embryo (i.e. blastocyst) can beused to derive a cell line capable of being maintained in tissue culturethat is referred to as embryonic stem (ES) cells. The use of ES cells toobtain hematopoietic populations of differentiated cells has beensuggested in Burkett et al., pp. 698-708. 1991, New Biologist, Vol. 3;Schmitt et al., pp. 728-740, 1991, Genes and Development, Vol. 5;Gutierrez-Ramos et al., pp. 9171-9175, 1992, Vol. 89; Keller et al., pp.473-486, Mol. Cell. Biol., Vol. 13; and Breier et al., pp. 521-532,1992, Development, Vol. 114. The use of ES cells to obtain endothelialpopulations of differentiated cells has been suggested by Wang et al.,pp. 303-316, 1992, Development, Vol. 114. Prior investigators, however,have failed to obtain populations of totipotent cells (i.e. cells thatcan develop into any lineage, discussed in detail below) and pluripotentcells (i.e. cells, that while unable to develop into all lineages ofcells, are at least able to develop into all hematopoietic lineages,also discussed in detail below). A reason for this failure is that theES cells were cultured under conditions in which the cells committed toa cellular lineage early in the tissue culture process. As a result,prior investigators failed to recognize a method for obtainingsubstantially homogeneous populations of totipotent or pluripotentembryonic cells that are useful for therapeutic or experimental use. Inaddition, prior investigators failed to recognize a method for inducingsubstantially homogeneous populations of totipotent or pluripotent cellsto develop into preferred cell types.

Thus, there remains a need to develop a population of cells that aretotipotent, pluripotent and precursor cells, and therefore, are capableof developing into a wide variety of cellular lineages.

SUMMARY OF THE INVENTION

The present invention relates to novel populations of precursor cellsthat are capable of developing into different cell types. The precursorcell populations of the present invention are particularly advantageousin that the populations can be maintained in tissue culture, andtherefore the cells are useful as a therapeutic reagent and a reagent toidentify compounds that control precursor cell growth anddifferentiation.

One embodiment of the present invention is cell population that includes(a) a HOX11 precursor cell population comprising cells having a cellsurface molecule FcγRII, FcγRIII, Thy-1, CD44, VLA-4α, LFA-1β orcombinations thereof; (b) a HOX11 precursor cell population comprisingcells having a cell surface molecule FcγRII, FcγRIII, CD44, VLA-4α,LFA-1β or combinations thereof; (c) a HOX11 precursor cell populationcomprising cells having a cell surface molecule HSA, CD44, VLA-4α,LFA-1β, ICAM-1 or combinations thereof; (d) a HOX11 precursor cellpopulation comprising cells having a cell surface molecule CD45, Aa4.1,Sca-1, HSA, FcγRII, FcγRIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α, LFA-1β orcombinations thereof; and (e) a HOX11 precursor cell populationcomprising cells having a cell surface molecule selected from the groupconsisting of CD45, Aa4.1, HSA, FcγRII, FcγRIII, Thy-1, Mac-1, Gr-1,CD44, VLA-4α, LFA-1β, ICAM-1 or combinations thereof. Such a precursorcell population includes cells of a mesodermal derived cellular lineage,more particularly of hematopoietic lineage, endothelial lineage, musclecell lineage, epithelial cell lineage and neural cell lineage. Alsoincluded in the present invention is a method to obtain a precursor cellpopulation of the present invention, such method further discussedbelow.

Another embodiment of the present invention is a method to identify aregulatory factor that influences the growth of a cell, comprising: (a)contacting a HOX11 precursor cell population with a regulatory factorselected from the group consisting of a putative regulatory factor, aknown regulatory factor and mixtures thereof; and (b) assessing theresponsiveness of the progenitor cell population to the regulatoryfactor. Preferred methods to assess the responsiveness of a progenitorcell population include performing an assay, such as a proliferationassay and/or a differentiation assay.

Yet another embodiment of the present invention is directed to a methodto identify a compound expressed during the development of a populationof embryonic stem cells, comprising characterizing at least a portion ofthe cellular composition of at least one cell contained in a HOX11progenitor cell population to identify a compound expressed during thedevelopment of a population of embryonic stem cells. Preferred compoundsto be identified comprise nucleic acids, proteins, carbohydrates andlipids, with cell surface molecules, secreted molecules, cytoplasmicsignal transduction molecules, and nucleic acid binding proteins beingparticularly preferred.

The present invention also includes a method to produce an antibody,comprising administering to an animal an effective amount of a proteinand/or peptide derived from a HOX11 progenitor cell population andrecovering an antibody capable of selectively binding to the protein.

The present invention also includes a method to identify a therapeuticreagent useful in the treatment of hematopoietic disorders, comprising:(a) contacting a HOX11 progenitor cell population with a compoundselected from the group consisting of a putative regulatory factor and aknown regulatory factor; and (b) assessing the responsiveness of theprogenitor cell population to the compound. In addition, the presentinvention includes a method to identify a neutralizing reagent,comprising: (a) contacting a HOX11 progenitor cell population with aknown regulatory factor to produce a controlled cell population; (b)combining the controlled cell population with a neutralizing reagent,which may include a known neutralizing compound of the regulatory factorand a putative neutralizing compound of the regulatory factor; and (c)assessing the responsiveness of the progenitor cell population to theneutralizing reagent.

The present invention also includes an endothelial cell populationproduced by the method comprising, (1) transforming an EB cellpopulation with a nucleic acid molecule encoding a Polyoma Middle Tantigen to form Polyoma Middle T EB cells; and (2) culturing the PolyomaMiddle T EB cells under conditions suitable to obtain an endothelialcell population. In particular, the present invention includes anendothelial cell population having the identifying characteristics ofD4T. The present invention also includes a method to identify ahematopoietic growth factor from such an endothelial cell population.

One embodiment of the present invention is a conditioned mediumcomprising a cell culture supernatant recovered from a culture of anendothelial cell population produced by the method comprising, (1)transforming an EB cell population with a nucleic acid molecule encodinga Polyoma Middle T antigen to form Polyoma Middle T EB cells; and (2)culturing the Polyoma Middle T EB cells under conditions suitable toobtain an endothelial cell population. The present invention alsoincludes various uses of a conditioned medium including, a method toidentify a hematopoietic cell growth factor and a method to expand andmature a population of precursor cells.

The present invention also includes an enhanced precursor cellpopulation, comprising a precursor cell population contacted with a cellculture supernatant recovered from a culture of an endothelial cellpopulation produced by the method comprising, (1) transforming an EBcell population with a nucleic acid molecule encoding a Polyoma Middle Tantigen to form Polyoma Middle T EB cells; and (2) culturing the PolyomaMiddle T EB cells under conditions suitable to obtain an endothelialcell population, wherein the precursor cell population, when contactedwith the cell supernatant results in the formation of an enhancedprecursor population.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one photograph in color.Copies of this patent with color photographs will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 is a schematic representation of the development of embryoniccell populations.

FIG. 2 shows a representative microscopic field of view of an embryoidbody population and an embryonic blast cell population.

FIG. 3 illustrates cell surface marker staining of an embryonic stemcell population, a Day 4 embryoid body population and a Day 6 embryonicblast cell population.

FIG. 4 shows a representative microscopic field of view of a mixedpopulation of erythroid and endothelial cells.

FIG. 5 illustrates the kinetics of embryonic blast cell populationdevelopment over time.

FIG. 6 is a schematic representation of a myeloid assay.

FIG. 7 illustrates the hematopoietic potential of embryonic blast cellpopulations.

FIG. 8 illustrates the effect of the age of embryoid body cellpopulations on multi-lineage embryonic blast cell populationdevelopment.

FIG. 9 illustrates a model of hematopoiesis during ontogeny.

FIG. 10 illustrates the effect of specific growth factors on embryonicblast cell population development.

FIG. 11 illustrates T cell receptor expression by an embryonic stemcell-derived T cell population.

FIG. 12 illustrates the effect of specific growth factors on T cellreceptor expression by an embryonic stem cell-derived T cell population.

FIG. 13 illustrates the effect of the age of an embryoid body cellpopulation on the development of a mixed population of erythroid andendothelial cells.

FIG. 14 illustrates the effect of specific growth factors on thedevelopment of a mixed population of erythroid and endothelial cells.

FIG. 15 illustrates the effect of the concentration of specific growthfactors on the development of a mixed population of erythroid andendothelial cells.

FIG. 16 shows representative microscopic fields of view of a mixedpopulation of erythroid and endothelial cells stained with vonWillebrand factor.

FIG. 17 illustrates the clonality of the development of a mixedpopulation of erythroid and endothelial cells.

FIG. 18 illustrates a schematic representation of the method used todevelop an immortalized HOX11 cell population.

FIG. 19 the factor responsiveness of an EBHX-1 cell population.

FIG. 20 illustrates the factor responsiveness of an EBHX-11 cellpopulation.

FIG. 21 illustrates the factor responsiveness of an EBHX-14 cellpopulation.

FIG. 22 illustrates the enhancement of BLAST cell growth when EB cellsare grown in the presence of D4T conditioned medium.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention includes populations of pluripotentand precursor cells that are capable of developing into differentcellular lineages when cultured under appropriate conditions. As usedherein, the term "population" refers to cells having the same ordifferent identifying characteristics. The term "lineage" refers to allof the stages of the development of a cell type, from the earliestprecursor cell to a completely mature cell (i.e. a specialized cell). Arepresentation of the developmental pathways of populations of embryoniccells of the present invention is shown in FIG. 1.

Referring to FIG. 1 and in accordance with the present invention, apopulation of totipotent embryonic stem (ES) cells are allowed todifferentiate and generate a population of pluripotent embryoid body(EB) cells in tissue culture. A population of pluripotent EB cells ofthe present invention can be dissociated and re-cultured to obtain twodistinct populations of cells depending on the growth factors present inthe culture medium. The first population includes pluripotent embryonicblast (BLAST) cells and the second population includes a mixedpopulation of endothelial and erythroid cells. According to the presentinvention, the term "growth factor" is used in its broadest context andrefers to all factors that are capable of stimulating the growth of acell, maintaining the survival of a cell and/or stimulating thedifferentiation of a cell.

The population of BLAST cells can be further cultured in the presence ofcertain growth factors to obtain a population of pre-lymphoid cells(i.e. cells capable of developing into lymphoid cells, such as T cellsand B cells) referred to herein as BLAST-LYM cells. The population ofblast cells can also be dissociated and re-cultured in the presence ofcertain growth factors to obtain a population of cells that includeserythrocytes and leukocytes other than lymphocytes. Cells in thispopulation are referred to herein as BLAST-NEM cells. The developmentalpotential of the foregoing populations of cells indicate that thepopulations represent early stages of differentiation.

A "precursor cell" can be any cell in a cell differentiation pathway(such as shown in FIG. 1) that is capable of differentiating into a moremature cell. As such, the term "precursor cell population" refers to agroup of cells capable of developing into a more mature cell. Aprecursor cell population can comprise cells that are totipotent, cellsthat are pluripotent and cells that are stem cell lineage restricted(i.e. cells capable of developing into less than all hematopoieticlineages, or into, for example, only cells of erythroid lineage). Asused herein, the term "totipotent cell" refers to a cell capable ofdeveloping into all lineages of cells. Similarly, the term "totipotentpopulation of cells" refers to a composition of cells capable ofdeveloping into all lineages of cells. Also as used herein, the term"pluripotent cell" refers to a cell capable of developing into a variety(albeit not all) lineages and are at least able to develop into allhematopoietic lineages (e.g., lymphoid, erythroid, and thrombocyticlineages). For example, a pluripotent cell can differ from a totipotentcell by having the ability to develop into all cell lineages exceptendothelial cells. A "pluripotent population of cells" refers to acomposition of cells capable of developing into less than all lineagesof cells but at least into all hematopoietic lineages. As such, atotipotent cell or composition of cells is less developed than apluripotent cell or compositions of cells. As used herein, the terms"develop", "differentiate" and "mature" all refer to the progression ofa cell from the stage of having the potential to differentiate into atleast two different cellular lineages to becoming a specialized cell.Such terms can be used interchangeably for the purposes of the presentapplication.

One aspect of the present invention is a method to produce a pluripotentpopulation of EB cells that, when cultured under appropriate conditions,are capable of developing into a variety of cell lineages, includingendothelial cell or hematopoietic lineage. A pluripotent EB cellpopulation of the present invention can be derived by culturing apopulation of totipotent ES cells in an embryoid body medium includingplatelet-poor fetal bovine serum (PP-FBS). According to the presentinvention, PP-FBS refers to fetal bovine serum not having inhibitors ofES cell differentiation (e.g., TGF-β). A preferred PP-FBS of the presentinvention comprises fetal bovine blood from which platelets have beenremoved and the resulting plasma has been clotted, thereby producingplatelet-poor serum. Suitable ES cells for use with the presentinvention include inner mass cells derived from an about 3.0 day old toabout 4.0 day old blastocyst, with a blastocyst about 3.5 days old beingmore preferred. ES cells of the present invention are derived from ananimal, preferably from a mammal, and more preferably from a human,mouse, primate, pig, cow, sheep, rabbit, rat, guinea pig or hamster.

In one embodiment, an EB cell population of the present invention isderived by culturing a population of ES cells in an embryoid bodymedium, which is medium that stimulates the differentiation of an EScell population to an EB cell population. Typically, an ES cellpopulation is maintained in an undifferentiated state by culturing thecells in an embryonic stem cell medium including leukemia inhibitoryfactor (LIF) and fetal calf serum (FCS). To produce an EB cellpopulation in accordance with the present invention, an ES cellpopulation is removed from the embryonic stem cell medium andre-cultured in embryoid body medium in which the LIF and the FCS havebeen replaced by either PP-FBS or normal FCS pre-selected for theability to promote EB cell development (referred to herein aspre-selected normal FCS). Both the absence of LIF and the presence ofPP-FBS or pre-selected normal FCS in the culture medium stimulates theES cell population to differentiate into an EB cell population of thepresent invention. An embryoid body medium of the present inventionincludes a suitable amount of PP-FBS or pre-selected normal FCS that iscapable of stimulating the differentiation of an ES cell population toan EB cell population. A preferred embryoid body cell medium of thepresent invention includes from about 5% to about 30%, more preferablyfrom about 10% to about 20%, and even more preferably about 15% PP-FBSor pre-selected normal FCS.

Applicants have discovered that culturing of an ES cell population for acertain period of time results in the differentiation of the ES cellpopulation to an EB cell population in which the EB cells arepluripotent. If cultured for too long, as has been done by priorinvestigators, the EB cell population loses pluripotency. As such, inaccordance with the present invention, an EB cell population of thepresent invention is derived by culturing a population of ES cells for asuitable amount of time to produce a pluripotent population of EB cells.In other words, an EB cell population of the present invention isderived by culturing a population of ES cells for an amount of time thatmaintains an EB cell population at a stage of pluripotency. Inparticular, the present invention includes a population of EB cells thatare derived by culturing a population of ES cells for a suitable amountof time to produce a population of EB cells that is capable ofdeveloping into an endothelial cell lineage and/or a hematopoietic celllineage. In accordance with the present invention, an EB cell populationis derived by culturing a population of ES cells from about 1 day toabout 7 days. A preferred EB cell population of the present invention isderived by culturing a population of ES cells from about 3 days to about4 days, with from about 72 hours to 96 hours being more preferred.

In accordance with the present invention, the culture conditions arealso important in obtaining a pluripotent EB cell population of thepresent invention from a totipotent population of ES cells. For example,an ES cell population is cultured in suspension to derive an EB cellpopulation. During culturing, variables such as cell density,temperature and CO₂ levels can be controlled to maximize the developmentof populations of EB cells. For example, it appears that the density ofcells in an ES cell culture can affect the development of an EB cellpopulation. While not being bound by theory, it is believed that ES cellpopulations produce one or more growth factors that are capable ofstimulating the differentiation of the ES cell population into an EBcell population. As such, the optimum cell density for the growth of anEB cell population is from about 1×10³ ES cells per ml to about 100×10³ES cells per ml, more preferably from about 2×10³ ES cells per ml toabout 10×10³ ES cells per ml, and even more preferably from about 3×10³ES cells per ml to about 4.5×10³ ES cells per ml. The optimumtemperature for the development of an EB cell population is from about35° C. and about 39° C., preferably from about 36° C. and 38° C., with atemperature of 37° C. being even more preferred. The optimum CO₂ levelsin the culturing environment for the development of EB cell populationsis from about 3% CO₂ to about 10% CO₂, more preferably from about 4% CO₂to about 6% CO₂, and even more preferably about 5% CO₂.

In a preferred embodiment, an EB cell population of the presentinvention is derived by culturing a population of ES cells in anembryoid body medium including Iscove's Modified Dulbecco's Medium(IMDM), with about 15% PP-FBS (obtained from Antech, Tex.),monothiolglycerol (MTG), transferrin, glutamine, at a cell density ofabout 4.5×10³ cells per ml of medium. The ES cell population is thencultured for about 96 hours, at about 37° C., in an about 5% CO₂-containing environment to obtain a population of pluripotent EB cells.

An EB cell population of the present invention is capable of developinginto cells of mesodermal cell lineage, of ectodermal cell lineage or ofendodermal cell lineage. As used herein, mesodermal cells include cellsof connective tissue, bone, cartilage, muscle, blood and blood vessel,lymphatic and lymphoid organ, notochord, pleura, pericardium,peritoneum, kidney and gonad. Ectodermal cells include epidermal tissuecells, such as those of nail, hair, glands of the skin, the nervoussystem, the external sense organs (e.g., eyes and ears) and mucousmembranes (such as those of the mouth and anus). Endodermal cellsinclude cells of the epithelium such as those of the pharynx,respiratory tract (except the nose), digestive tract, bladder andurethra cells. Preferred cells within an EB cell population of thepresent invention include cells of at least one of the followingcellular lineages: hematopoietic cell lineage, endothelial cell lineage,epithelial cell lineage, muscle cell lineage and neural cell lineage.More preferred cells within an EB cell population of the presentinvention include cells of erythroid lineage, endothelial lineage,leukocyte lineage and thrombocyte lineage. Even more preferred cellswithin an EB cell population of the present invention include cells oferythroid lineage (including primitive and definitive erythroidlineages), macrophage lineage, neutrophil lineage, mast cell lineage,megakaryocyte lineage, natural killer cell lineage, eosinophil lineage,T cell lineage, endothelial cell lineage and B cell lineage.

An EB cell population of the present invention includes a colony ofcells having substantially the same morphology as the colony of cellsshown in FIG. 2, cell colony A. The EB cell population shown in FIG. 2,cell colony A, was obtained when ES cells were grown as described inExample 1. Referring to FIG. 2, the EB cell population shown as cellcolony A has a general morphology of tightly packed cells, in whichindividual cells are not easily detectable.

An EB cell population of the present invention that has been derived byculturing a population of ES cells for 4 days (i.e. Day 4 EB) caninclude cells that have substantially the same antibody staining patternas shown in FIG. 3A and 3B, when such EB cells are stained according tothe method described in Example 2. Referring to FIG. 3A and 3B, a Day 4EB cell population expresses substantially low amounts of Sca-1, C-kitreceptor and Class I H-2b, and essentially no Thy 1, VLA-4, CD44 andCD45.

Another aspect of the present invention is a method to produce a celltype, such as a mesodermal cell, an ectodermal cell and/or an endodermalcell that includes the steps of: (a) selecting a desired cell type toproduce; and (b) culturing an embryoid body cell population of thepresent invention under conditions suitable to obtain the desired celltype. Suitable culture conditions for obtaining a desired cell typeinclude culturing the EB cell population in a medium including a growthfactor that is able to stimulate the EB cell population to differentiateto the desired cell type(s). As used herein, the term "a" refers to "atleast one", or "one or more." For example, an EB cell population can becultured in a medium including a growth factor capable of promoting thedifferentiation of the cell population into a hematopoietic cell type. Apreferred culture condition for obtaining a desired cell type thatincludes erythroid and endothelial cells includes culturing an EB cellpopulation of the present invention in the presence of erythropoietin(EPO) and vascular endothelial growth factor (VEGF; described in detailbelow). Another preferred culture condition for obtaining a desired celltype of embryonic blast cells includes culturing an EB cell populationof the present invention in the presence of C-kit ligand, interleukin 1(IL-1), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 11(IL-11), EPO, VEGF, and mixtures thereof, to obtain a cell of aparticular cell type (described in detail below).

In the mouse, the first visible signs of blood cell development are theappearance of foci or red blood cells in a tissue called the yolk sac at7.5 days of gestation. These early appearing red blood cells areprimitive erythroid cells. Cells which are destined to make bloodvessels, the endothelial cells, appear at almost the same time and inthe same location as such embryonic red blood cells. Without being boundby theory, it is believed that the parallel appearance of the two typesof cells in close proximity indicates that the two cell types developfrom a common precursor. Prior investigators, however, have merelytheorized about the existence of such a precursor cell population andhave not taught or enabled the isolation of the population. Applicantshave identified for the first time a population of precursor cells thatare able to develop into endothelial and erythroid lineages.

One aspect of the present invention is a method to produce a mixedpopulation of erythroid and endothelial cells by culturing a populationof pluripotent EB cells of the present invention in anendothelial/erythroid cell medium, which is medium that stimulates thedifferentiation of an EB cell population of the present invention to apopulation of erythroid and endothelial cells. An endothelial/erythroidcell medium of the present invention includes a suitable amount of agrowth factor capable of stimulating the development of an EB cellpopulation into a mixed population of endothelial cells and erythroidcells. A preferred endothelial/erythroid cell medium of the presentinvention includes a hematopoietic cell growth factor, an endothelialcell growth factor, homologues of such growth factors, or mixtures ofsuch growth factors and/or homologues. A more preferredendothelial/erythroid cell medium of the present invention includesC-kit ligand, EPO and VEGF, homologues of such growth factors, ormixtures of such growth factors and/or homologues. An even morepreferred cell medium of this embodiment of the present inventionincludes EPO and VEGF.

According to the present invention, an endothelial/erythroid cell mediumof the present invention includes a suitable growth factor, and PP-FBSor pre-selected normal FCS. A preferred endothelial/erythroid cellmedium of the present invention includes from about 5% to about 30%,more preferably from about 7% to about 20%, and even more preferablyabout 10% PP-FBS or pre-selected normal FCS.

Also according to the present invention, an EB cell population of thepresent invention is preferably cultured in methyl cellulose to obtain amixed population of endothelial and erythroid cells. A suitable amountof methyl cellulose for culturing EB cell populations is an amount thatenables the EB cells to associate as groups (i.e. clumps or clusters) ofcells, thereby stimulating growth and/or differentiation of the EB cellsinto cells. A preferred amount of methyl cellulose in which to culturean EB cell population of the present invention to obtain a mixedpopulation of endothelial and erythroid cells is from about 0.25% toabout 2%, more preferably from about 0.5% to about 1.5%, and even morepreferably at about 1%.

Also according to the present invention, an EB cell population of thepresent invention is cultured in an endothelial/erythroid cell mediumfor a sufficient amount of time to allow the EB cell population todifferentiate to a mixed population of endothelial and erythroid cells.A preferred amount of time to culture an EB cell population is fromabout 5 days to about 12 days. A more preferred amount of time toculture an EB cell population is from about 6 days to about 11 days. Aneven more preferred amount of time to culture an EB cell population isfrom about 7 days to about 10 days.

Other culture conditions (i.e. in addition to time and medium) which caneffect the development of a mixed population of endothelial anderythroid cells of the present invention from an EB cell population ofthe present invention includes the temperature and CO₂ content of theculture environment. The optimum temperature for the development of amixed population of endothelial and erythroid cells of the presentinvention is from about 35° C. to about 39° C., preferably from about36° C. to 38° C., with a temperature of 37° C. being even morepreferred. The optimum CO₂ levels in the culturing environment for thedevelopment of a mixed population of endothelial and erythroid cells isfrom about 3% CO₂ to about 10% CO₂, more preferably from about 4% CO₂ toabout 6% CO₂, and even more preferably about 5% CO₂.

A mixed population of endothelial and erythroid cells of the presentinvention is derived by culturing a population of EB cells at a suitablecell density to produce a mixed population of endothelial and erythroidcells. The optimum cell density for the growth of the population ispreferably from about 5×10⁴ cells to about 7.5×10⁵ EB cells, morepreferably from about 1.5×10⁵ cells to about 6×10⁵ EB cells, and evenmore preferably from about 2×10⁵ cells to about 5×10⁵ EB cells per ml ofculture medium.

In a preferred embodiment, a mixed population of endothelial anderythroid cells of the present invention is derived by culturing apopulation of EB cells of the present invention in anendothelial/erythroid cell medium including IMDM, with about 10% PP-FBS,1% methyl cellulose, and a mixture of growth factors including VEGF andEPO for about 7 days, at about 37° C., in an about 5% CO₂ -containingenvironment to obtain a mixed population of endothelial and erythroidcells.

In one embodiment, a mixed population of endothelial and erythroid cellsincludes one or more cells of endothelial lineage or erythroid lineage.A preferred mixed population of endothelial and erythroid cells includesone or more cells that can be stained with von Willebrand factoraccording to the method described in Example 10, and/or one or morecells that can absorb diI-acetylated-low density lipoproteins whenlabelled according to the method described in Example 10.

A mixed population of endothelial and erythroid cells of the presentinvention includes one or more cells having a substantially similarmorphology as the cells shown in FIG. 4. Referring to FIG. 4, generally3 types of cells having different morphologies can be found in a mixedpopulation of endothelial and erythroid cells of the present invention.A first cell type, indicated as cell A in FIG. 4, comprises an erythroidcell having the typical characteristics of a distinct compact cluster ofsmall cells having red color. A second cell type, indicated as cell B inFIG. 4, comprises a spherical cell having a larger size than anerythroid cell, such as cell A. A third cell type, indicated as cell Cin FIG. 4, comprises a spherical cell having a similar size as anerythroid cell but having a single long process extending from the cell.According to the present invention, both the second cell type (i.e. cellB in FIG. 4) and the third cell type (i.e. cell C in FIG. 4) can bestained with von Willebrand factor and can absorb diI-acetylated-lowdensity lipoproteins, thereby indicating that such cell types arerepresentative of endothelial cells.

The present invention also includes an endothelial cell population madeby the method comprising: (1) transforming an EB cell population of thepresent invention with a nucleic acid molecule encoding a Polyoma MiddleT antigen to form Polyoma Middle T EB cells; and (2) culturing thePolyoma Middle T EB cells under conditions suitable to obtain anendothelial cell population. A preferred EB cell population comprises anES cell population cultured for no more than about 8 days, preferablyfrom about 2 days to about 8 days. A more preferred EB cell populationcomprises an ES cell population cultured for no more than about 6 days,preferably from about 3 days to about 6 days. An even more preferred EBcell population comprises an ES cell population cultured for about 4days, using the culture conditions for EB cell formation that aredescribed in detail herein.

Methods for transformation and expression of Polyoma Middle T antigen inan EB cell population of the present invention include subcloning anucleic acid molecule encoding Polyoma Middle T antigen into aretroviral vector, producing virus and infecting an EB cell populationwith the retrovirus using methods standard to those in the art. Apreferred nucleic acid molecule encoding Polyoma Middle T antigencomprises a full-length Polyoma Middle T antigen gene as disclosed inWilliams et al. (Cell 52:121-131, 1988). A preferred retroviral vectorof the present invention comprises an N2 vector (described in Keller etal., Nature 318:149-154, 1985).

According to the present invention, a suitable culture condition forobtaining an endothelial cell population of the present inventionincludes culturing Polyoma Middle T antigen transformed EB cells inculture medium comprising one or more growth factors as disclosedherein. A more preferred culture medium comprises endothelial cellgrowth supplement.

In accordance with the present invention, the medium also includesnormal FCS, in addition to one or more growth factors described above. Apreferred concentration of normal FCS to include in the medium of thepresent invention includes from about 10% and about 25%, more preferablyfrom about 12% to about 20%, and even more preferably about 10% normalFCS.

Other culture conditions (i.e. in addition to time and medium) which caneffect the development of an endothelial population of the presentinvention include the temperature and CO₂ content of the cultureenvironment as disclosed in detail herein.

In a preferred embodiment, an endothelial cell population of the presentinvention is derived by culturing a population of Polyoma Middle Tantigen transformed EB cells of the present invention in a mediumincluding IMDM, with about 10% normal FCS, and endothelial cell growthsupplement. The transformed EB cell population is grown a cell densityof from about 1×10⁵ cells per ml of medium to about 5×10⁵ cells per mlof medium for about 2 months at about 37° C., in an about 5% CO₂-containing environment.

An endothelial cell population derived by the foregoing method includescells expressing cell surface markers characteristic of endothelialcells. In particular, an endothelial cell population comprises cellsexpressing Flk-1 and/or CD31. In addition, an endothelial cellpopulation comprises cells that can absorb diI-acetylated-low densitylipoproteins.

In a preferred embodiment, an endothelial cell population of the presentinvention includes a cell population having the identifyingcharacteristics of D4T (described in detail in Example 18).

It is within the scope of the present invention that a retrovirallytransformed endothelial cell of the present invention can be used toidentify one or more known and/or unknown compounds contained in theconditioned medium that are useful for enhancing a cell population ofthe present invention. As used herein, the term "enhancing" refers toincreasing the growth and/or the differentiation (i.e., maturation) of acell population in the presence compared with in the absence of acompound. Preferred compounds to identify using a retrovirallytransformed endothelial cell population of the present inventioninclude: (1) unknown compounds having hematopoietic growth factoractivity, either alone or in combination with a known hematopoieticgrowth factor; and (2) unknown factors that induce pre-hematopoieticcells to develop into hematopoietic cells. Such compounds can beidentified using any method standard in the art. For example, RNAexpression in the cells can be analyzed for the presence or absence ofRNA transcripts encoding known compounds by using probes specific forthe nucleotide sequence of such compounds. In addition, standardexpression cloning techniques (as described in Sambrook et al., ibid.)to identify nucleic acid sequences encoding both known and unknowncompounds.

Another aspect of the present invention is a method to produce apluripotent population of BLAST cells that, while unable to develop intoall lineages of cells, are at least able to develop into allhematopoietic lineages, when cultured under appropriate conditions. Inone embodiment, a pluripotent BLAST cell population of the presentinvention can be derived by culturing a population of pluripotent EBcells of the present invention in an embryonic blast cell medium, whichis medium that stimulates the differentiation of an EB cell populationof the present invention to a BLAST cell population of the presentinvention. An embryonic blast cell medium of the present inventionincludes a suitable amount of a growth factor capable of stimulating thedevelopment of an EB cell population into a pluripotent BLAST cellpopulation. A preferred embryonic blast cell medium of the presentinvention includes a hematopoietic cell growth factor, an endothelialcell growth factor or a mixture thereof. As used herein, a homologue ofa specific growth factor refers to a compound that is capable of havingsubstantially similar activity as that growth factor; i.e. a homologueof a growth factor is substantially similar to that growth factor. Forexample, a homologue of a specific growth factor can bind to the cellsurface receptor of that growth factor in such a manner that the surfacereceptor is stimulated to induce an appropriate cellular functionsimilar to that effected by the specific growth factor. A more preferredembryonic blast cell medium of the present invention includes C-kitligand, IL-1, IL-3, IL-6, IL-11, EPO, VEGF, homologues of such growthfactors, or mixtures of such growth factors and/or homologues. An evenmore preferred embryonic blast cell medium of the present inventionincludes a mixture of C-kit ligand, IL-1, IL-6 and IL-11; a mixture ofC-kit ligand, EPO and VEGF; or C-kit ligand alone.

In accordance with the present invention, an embryonic blast cell mediumincludes PP-FBS or pre-selected normal FCS in addition to one or moregrowth factors described above. A preferred embryonic blast cell mediumof the present invention includes from about 5% and about 20%, morepreferably from about 7% to about 15%, and even more preferably about10% PP-FBS or pre-selected normal FCS.

Also according to the present invention, an EB cell population of thepresent invention is cultured in methyl cellulose to obtain a populationof BLAST cells of the present invention. A suitable amount of methylcellulose for culturing EB cell populations is an amount that enablesthe EB cells to associate as groups (i.e. clumps or clusters) of cells,thereby stimulating growth and/or differentiation of the EB cells intoBLAST cells. A preferred amount of methyl cellulose in which to culturean EB cell population of the present invention is from about 0.25% toabout 2.0%, more preferably from about 0.5% to about 1.5%, and even morepreferably at about 1%.

A BLAST cell population of the present invention is derived by culturinga population of EB cells at a suitable cell density to produce apluripotent population of BLAST cells. The optimum cell density for thegrowth of a BLAST cell population is preferably from about 1×10⁵ cellsto about 7.5×10⁵ EB cells, more preferably from about 1.5×10⁵ cells toabout 6×10⁵ EB cells, and even more preferably from about 2×10⁵ cells toabout 5×10⁵ EB cells per ml of culture medium.

Applicants have discovered that culturing of an EB cell population for acertain period of time in accordance with the present invention resultsin the differentiation of an EB cell population to a BLAST cellpopulation in which the BLAST cells are pluripotent. If cultured for toolong, as has been done by prior investigators, the BLAST cell populationloses pluripotency. As such, a BLAST cell population of the presentinvention is derived by culturing a population of EB cells for asuitable amount of time to produce a pluripotent population of BLASTcells. In particular, a population of EB cells are cultured for asuitable amount of time to produce a population of BLAST cells that iscapable of developing into any hematopoietic cell lineage. In otherwords, a BLAST cell population of the present invention is derived byculturing a population of EB cells for an amount of time that maintainsa BLAST cell population at a stage of pluripotency. A preferred BLASTcell population is derived by culturing a population of EB cells from atleast about 2 days to about 15 days. A more preferred BLAST cellpopulation of the present invention is derived by culturing a populationof EB cells from about 3 days to about 10 days. An even more preferredBLAST cell population of the present invention is derived by culturing apopulation of EB cells from about 3 days to about 6 days.

Other culture conditions (i.e. in addition to time and medium) which caneffect the development of a BLAST cell population of the presentinvention from an EB cell population of the present invention includethe temperature and CO₂ content of the culture environment. The optimumtemperature for the development of a BLAST cell population of thepresent invention is from about 35° C. to about 39° C., preferably fromabout 36° C. to about 38° C., with a temperature of about 37° C. beingeven more preferred. The optimum CO₂ levels in the culturing environmentfor the development of BLAST cell populations is from about 3% CO₂ toabout 10% CO₂, more preferably from about 4% CO₂ to about 6% CO₂, andeven more preferably about 5% CO₂.

In a preferred embodiment, a BLAST cell population of the presentinvention is derived by culturing a population of EB cells of thepresent invention in an embryonic blast cell medium including IMDM, withabout 10% PP-FBS, 1% methyl cellulose, and either a mixture of growthfactors including IL-1, IL-6, IL-11, C-kit ligand, EPO and VEGF, orC-kit ligand alone. The EB cell population is grown a cell density offrom about 2×10⁵ cells per ml of medium to about 5×10⁵ cells per ml ofmedium. After reaching that density, the EB cell population is thencultured for about 6 days, at about 37° C., in an about 5% CO₂-containing environment to obtain a population of pluripotent BLASTcells.

In another preferred embodiment, an EB cell population of the presentinvention is grown in a culture medium comprising conditioned mediumderived from the supernatant of endothelial cell cultures. According tothe present invention, endothelial cell conditioned medium is producedby culturing an endothelial cell population of the present invention ina culture medium suitable for the growth of an endothelial cellpopulation until the cells become confluent in the culture dish. Thesupernatant from the cultures are recovered using methods standard inthe art to obtain conditioned medium.

A suitable endothelial cell population useful for the production ofconditioned medium includes an endothelial cell population derived froman EB cell population of the present invention. Preferably, aconditioned medium of the present invention is derived from anendothelial cell population produced using a retrovirally transformed EBcell population of the present invention. A particularly preferredendothelial cell population useful for the production of conditionedmedium includes a cell population having the identifying characteristicsof a D4T cell population.

Suitable medium for the growth of endothelial cells includes culturemedium comprising one or more growth factors described herein,preferably, an endothelial cell growth supplement (ECGF). An endothelialcell culture medium preferably comprises from about 12 μg/ml to about300 μg/ml, more preferably from about 25 μg/ml to about 200 μg/ml andeven more preferably from about 50 μg/ml to about 100 μg/ml ECGF.

A preferred conditioned medium of the present invention, when added to a3 day EB cell population, is capable of enhancing (i.e. stimulating thegrowth and/or differentiation) of a BLAST cell population of the presentinvention about 2-fold when a conditioned medium is added to a cultureof about 5×10⁴ cells from a 3 day EB cell population, about 5-fold whena conditioned medium is added to a culture of about 1.7×10⁴ cells from a3 day EB cell population and about 23-fold when a conditioned medium isadded to a culture of about 6×10³ cells from a 3 day EB cell population,when the culture is performed under conditions suitable for thedevelopment of a BLAST cell population. Preferably, the EB cellpopulation culture medium comprises VEGF, EPO, C-kit ligand or mixturesthereof.

In a preferred embodiment, a conditioned medium of the present inventionincludes medium recovered from about 72 hour cultures of D4T cells grownin culture medium comprising ECGF. It is within the scope of the presentinvention that a characteristic of a D4T cell population is the abilityto produce compounds that condition a medium in such a manner that theconditioned medium is capable of enhancing a BLAST cell population inthe manner disclosed herein.

It is within the scope of the present invention that a conditionedmedium of the present invention can be used to identify one or moreknown and/or unknown compounds contained in the conditioned medium thatare useful for enhancing the growth of a cell population of the presentinvention. Preferred compounds to identify using a conditioned medium ofthe present invention include: (1) unknown compounds havinghematopoietic growth factor activity, either alone or in combinationwith a known hematopoietic growth factor; and (2) unknown factors thatinduce pre-hematopoietic cells to develop into hematopoietic cells. Suchcompounds can be identified using any method standard in the art. Forexample, immunoassays can be used to identify the presence of knowncompounds in a conditioned medium of the present invention.Alternatively, standard biochemical protein separation techniques (e.g.,antibody binding studies, gel electrophoresis and various chromatographytechniques, in particular HPLC, known to those of skill in the art) canbe used to identify and isolate individual or families of proteins froma conditioned medium. The ability of an unknown compound to effect cellgrowth can be tested using, for example, the method described in Example18. Various types of cell growth assays are applicable in this situationand that any cell population of the present invention can be employed insuch assays.

It is also within the scope of the present invention that a conditionedmedium of the present invention can be used to enhance precursorpopulations of cells, preferably human hematopoietic precursor cells. Assuch, a conditioned medium of the present invention is capable ofenhancing the growth and/or differentiation of a cell populationincluding totipotent, pluripotent and/or stem cell lineage restrictedcells. Such cells include, for example, fetal, embryonic and adult organcells. Enhancement of precursor populations of cells is particularlyuseful in the treatment of diseases that involve replenishing precursorcell populations in a subject. For example, cancer patients undergoingchemotherapy, radiotherapy and/or bone marrow transplants are preferredrecipients of precursor cell populations enhanced using a conditionedmedium of the present invention.

Precursor cell populations can be enhanced by culturing such cells undersuitable culture conditions in the presence of an effective amount ofconditioned medium. One can determine the culture conditions and amountof conditioned medium to used based upon parameters, such as the celltype being expanded, the health of the cells being expanded and theextent of expansion required.

The scope of the invention also includes an enhanced precursor cellpopulation, comprising a precursor cell population (i.e. a population ofcells comprising precursor cells) contacted with a conditioned medium ofthe present invention, wherein the step of contacting results in theformation of an enhanced precursor population. Preferably, an enhancedprecursor cell population comprises about 2-fold, more preferably about5-fold and even more preferably about 20-fold more cells than theprecursor cell population. A particularly preferred precursor cellpopulation comprises a human precursor cell population.

Being pluripotent, a BLAST cell population of the present inventionincludes cells of hematopoietic and other cell lineages. In particular,a BLAST cell population of the present invention includes cells oferythroid lineage, endothelial lineage, leukocyte lineage andthrombocyte lineage. A preferred BLAST cell population of the presentinvention includes cells capable of developing into primitive erythroidcells, definitive erythroid cells, macrophages, neutrophils, mast cells,T cells, endothelial cell, B cells, natural killer cells,megakaryocytes, eosinophils, and progenitors and progeny thereof. Asused herein, a primitive erythroid cell is characterized by the cell'snucleated morphology and expression of embryonic globin. A definitiveerythroid cell (also referred to as an adult erythroid cell) ischaracterized by the cell's expression of adult globin and eventualenucleation. As used herein, a "progenitor" cell refers to an ancestorof a cell (i.e. a cell from which a subject cell is derived). As usedherein, a "progeny" cell refers to a cell derived from a subject cell.

A BLAST cell population of the present invention includes a colony ofcells having substantially the same morphology as the colony of cellsshown in FIG. 2, cell colony B. The BLAST cell population shown in FIG.2, cell colony B was obtained when EB cells were grown as described inExample 3. Referring to FIG. 2, the BLAST cell population shown as cellcolony B has a general morphology of clumped, but not tightly packedcells, in which individual cells can be discerned when the colony isviewed under the microscope.

A BLAST cell population of the present invention that has been derivedby culturing a population of EB cells for 6 days (i.e. Day 6 BLASTS) caninclude cells that have substantially the same antibody staining patternas shown in FIG. 3A and 3B, when such BLAST cells are stained accordingto the method described in Example 4. Referring to FIG. 3A and 3B, a Day6 BLAST cell population expresses substantial amounts of CD44, C-kitreceptor, Sca-1 and VLA-4, and essentially no Class I H-2b, Thy 1 andCD45.

Another aspect of the present invention is a method to produce apopulation of BLAST-LYM cells that are able to develop into a cell oflymphoid lineage, when cultured under appropriate conditions. In oneembodiment, a BLAST-LYM cell population of the present invention can bederived by culturing a population of pluripotent BLAST cells of thepresent invention in a BLAST-LYM cell medium, which is medium thatstimulates the differentiation of a BLAST cell population of the presentinvention to a BLAST-LYM cell population of the present invention. ABLAST-LYM cell medium of the present invention includes a suitableamount of one or more lymphoid cell growth factors that are capable ofstimulating the differentiation of a BLAST cell population to anBLAST-LYM cell population. A preferred BLAST-LYM cell medium for theproduction of BLAST-LYM cells of the present invention includes one ormore of the lymphoid growth factors IL-7, C-kit ligand, insulin-likegrowth factor 1 (IGF-1), VEGF, EPO, a growth factor produced by anembryoid body cell, homologues of such growth factors, or mixtures ofsuch growth factors and/or homologues. A more preferred BLAST-LYM cellmedium for the production of BLAST-LYM cells of the present inventionincludes one or more of the lymphoid growth factors IL-7, C-kit ligand,insulin-like growth factor 1 (IGF-1), homologues of such growth factors,or mixtures of such growth factors and/or homologues.

According to the present invention, a BLAST-LYM cell medium of thepresent invention includes PP-FBS or pre-selected normal FCS as well asone or more suitable growth factor as described above. A preferredBLAST-LYM cell medium of the present invention includes from about 5% toabout 30%, more preferably from about 7% to about 20%, and even morepreferably about 10% PP-FBS or pre-selected normal FCS.

Also according to the present invention, a BLAST cell population of thepresent invention is preferably cultured in methyl cellulose to obtain apopulation of BLAST-LYM cells. A suitable amount of methyl cellulose forculturing BLAST cell populations is an amount that enables the BLAST-LYMcells to associate as groups (i.e. clumps or clusters) of cells, therebystimulating growth and/or differentiation of the BLAST cells intoBLAST-LYM cells. A preferred amount of methyl cellulose in which toculture a BLAST cell population of the present invention to obtain aBLAST-LYM is from about 0.25% to about 2.0%, more preferably from about0.5% to about 1.5%, and even more preferably at about 1%.

A BLAST-LYM cell population of the present invention is derived byculturing a population of BLAST cells for a suitable amount of time toproduce a BLAST-LYM cell population able to develop into a lymphoidlineage. In particular, the present invention includes a population ofBLAST-LYM cells that are derived by culturing a population of BLASTcells for a suitable amount of time to produce a population of BLAST-LYMcells that are capable of developing into a T cell or a B cell whencultured under appropriate conditions. According to the presentinvention, a T cell includes a cell that represents a given stage of Tcell maturation, and as such, can rearrange from immature T cells havingrearranged T cell receptor germline genes to mature T cells expressingαβ T cell receptor proteins. Also according to the present invention, aB cell can include a cell that represents a given stage of B cellmaturation, and as such, can range from an early B cell havingrearranged diversity (i.e. D) region and joining (i.e. J) regionimmunoglobulin germline genes, more preferably a cell having rearrangeddiversity D, J and variable (i.e. V) region immunoglobulin germlinegenes, to a plasma cell that is able to secrete immunoglobulin proteins.A BLAST-LYM cell population is derived by culturing a population ofBLAST cells from about 3 day to about 10 days, preferably for about 6days.

In accordance with the present invention, other culture conditions (i.e.in addition to time and medium) are also important in obtaining aBLAST-LYM cell population of the present invention from a population ofBLAST cells. During culturing, variables such as cell density,temperature and CO₂ levels can be controlled to maximize the developmentof populations of BLAST-LYM cells. For example, it appears that thedensity of cells in a BLAST cell culture can affect the development of aBLAST-LYM cell population. The optimum cell density for the growth of anBLAST-LYM cell population is from about 5×10⁴ BLAST cells per ml toabout 7.5×10⁵ BLAST cells per ml, more preferably from about 1×10⁵ BLASTcells per ml to about 6×10⁵ BLAST cells per ml, and even more preferablyfrom about 2.5×10⁵ BLAST cells per ml to about 5×10⁵ BLAST cells per ml.The optimum temperature for the development of an BLAST-LYM cellpopulation is from about 35° C. to about 39° C., preferably from about36° C. to 38° C., with a temperature of 37° C. being even morepreferred. The optimum CO₂ levels in the culturing environment for thedevelopment of BLAST-LYM cell populations is from about 3% CO₂ to about10% CO₂, more preferably from about 4% CO₂ to about 6% CO₂, and evenmore preferably about 5% CO₂.

In a preferred embodiment, a BLAST-LYM cell population of the presentinvention is derived by culturing an individual BLAST cell colony in amedium including IMDM, with about 10% PP-FBS, 1% methyl cellulose, and amixture of growth factors including IL-7, IGF-1 and C-kit ligand forabout 6 days at about 37° C., in an about 5% CO₂ -containing environmentto obtain a population of BLAST-LYM cells.

In accordance with the present invention, a BLAST-LYM cell population ofthe present invention includes cells of a lymphoid lineage. Preferredcells within a BLAST-LYM cell population of the present inventioninclude cells of a T cell lineage, a B cell lineage, and/or a naturalkiller cell lineage. More preferred cells within a BLAST-LYM cellpopulation of the present invention can develop into T cells havingrearranged T cell receptor germline genes and B cells having rearrangedD and J region immunoglobulin germline genes. Particularly preferredcells within a BLAST-LYM cell population of the present invention candevelop into a T cell expressing T cell receptor proteins, such as αβand/or γδ T cell receptors, or a B cell having rearranged V, D and Jregion immunoglobulin germline genes.

One aspect of the present invention is a method to produce a lymphoidcell population that includes the steps of: (a) culturing a BLAST cellpopulation in an BLAST-LYM cell medium including one or more lymphoidcell growth factors to produce a BLAST-LYM cell population; and (b)culturing the BLAST-LYM cell population with cells selected from thegroup consisting of fetal thymi culture cells and bone marrow stromalcells to obtain a lymphoid cell population.

In one embodiment, a BLAST-LYM cell population of the present inventionis cultured in a fetal thymi culture to obtain a population of T cells.Preferably, a BLAST cell population cultured in the presence of C-kitligand, IGF-1 and IL-7 is used to produce a BLAST-LYM cell which iscultured in a fetal thymi culture to produce a T cell population.Techniques to perform fetal thymi cultures are well known to those ofskill in the art. Preferably, a BLAST-LYM population is cultured in afetal thymi culture for from about 1 week to about 6 weeks, morepreferably for from about 1.5 weeks to about 4 weeks, and even morepreferably for from about 2 weeks to about 3 weeks. A preferred T cellpopulation of the present invention is a population of cells comprisingfrom about 1% to about 75% T cells, more preferably from about 3% toabout 65% T cells, and even more preferably from about 5% to about 50% Tcells. It is within the scope of the invention, however, that a T cellpopulation can also include other lymphocyte subpopulations that aretypically found in a thymic population. For example, a T cell populationof the present invention can include macrophages, dendritic cells,natural killer cells and epithelial cells. The T cells included in a Tcell population of the present invention preferably include T cellshaving rearranged T cell receptor germline genes and more preferablyinclude T cells expressing T cell receptor proteins, including αβ and/orγδ T cell receptors.

In another embodiment, a BLAST-LYM cell population of the presentinvention is cultured in the presence of bone marrow stromal cells toobtain a population of B cells. Preferably, a BLAST cell populationcultured in the presence of C-kit ligand alone is used to produce aBLAST-LYM cell which is cultured with bone marrow stromal cells toproduce a B cell population. Techniques to perform bone marrow stromalcell cultures are well known to those of skill in the art. Preferably, aBLAST-LYM population is cultured in the presence of bone marrow stromalcells for from about 3 days to about 75 days, more preferably for fromabout 7 days to about 45 days, and even more preferably for from about14 days to about 21 days. A preferred B cell population of the presentinvention is a population of cells comprising from about 0.5% to about20% B cells, more preferably from about 0.75% to about 17% B cells, andeven more preferably from about 1% to about 15% B cells. A preferred Bcell population includes B cells having rearranged D and J regionimmunoglobulin germline genes, and more preferably B cells havingrearranged V, D and J region germline genes.

Another aspect of the present invention is a method to produce apopulation of BLAST-NEM cells that are able to develop into a cell ofcertain hematopoietic lineages when cultured under appropriateconditions. According to the present invention, a cell of hematopoieticlineage is able to develop into erythrocyte cells (i.e. a red bloodcell), certain leukocyte cells (i.e. a white blood cell other thanlymphocytes), or thrombocyte cells (i.e. platelet cell). Leukocyte cellsinclude granular leukocytes, including eosinophils, basophils,neutrophils, and mast cells; as well as non-granular leukocytes,including megakaryocytes, polymorphonuclear cells, lymphocytes andmonocytes (i.e. macrophages). A BLAST-NEM cell population of the presentinvention comprises cells that are able to develop into anyhematopoietic cell type other than lymphocytes. A preferred BLAST-NEMcell population of the present invention includes cells that are able todevelop into erythrocytes, leukocytes other than lymphocytes, orthrombocytes. A more preferred BLAST-NEM cell population of the presentinvention includes cells that are able to develop into primitiveerythroid cells, definitive erythroid cells, macrophages, mast cells,neutrophils, eosinophils, megakaryocytes, undifferentiated hematopoieticcell colonies, or progenitors or progeny thereof. An even more preferredBLAST-NEM cell population of the present invention includes cells thatare able to develop into primitive erythroid cells, definitive erythroidcells, macrophages, mast cells, neutrophils, or progenitors or progenythereof.

In one embodiment, a BLAST-NEM cell population of the present inventioncan be derived by culturing a population of pluripotent BLAST cells ofthe present invention in a BLAST-NEM cell medium, which is a medium thatstimulates the differentiation of a BLAST cell population of the presentinvention to a BLAST-NEM cell population of the present invention. AnBLAST-NEM cell medium of the present invention includes a suitableamount of one or more BLAST-NEM cell growth factors that are capable ofstimulating the differentiation of a BLAST cell population to anBLAST-NEM cell population. For purposes of this application, BLAST-NEMcell growth factors differ from hematopoietic growth factors in thathematopoietic growth factors of the present invention include lymphoidfactors and the BLAST-NEM cell growth factors of the present inventiondo not. A preferred BLAST-NEM cell medium includes one or more of theBLAST-NEM cell growth factors C-kit ligand, IL-1, IL-3, IL-6, IL-11,VEGF, EPO, homologues of such growth factors, or mixtures of such growthfactors and/or homologues. A more preferred BLAST-NEM cell mediumincludes C-kit ligand, IL-1, IL-3, IL-6, IL-11, VEGF and EPO.

According to the present invention, a BLAST-NEM cell medium of thepresent invention includes PP-FBS or pre-selected normal FCS as well asone or more suitable growth factor as described above. A preferredBLAST-NEM cell medium of the present invention includes from about 5% toabout 30%, more preferably from about 7% to about 20%, and even morepreferably about 10% PP-FBS or pre-selected normal FCS.

Also according to the present invention, a BLAST cell population of thepresent invention is preferably cultured in methyl cellulose to obtain apopulation of BLAST-NEM cells. A suitable amount of methyl cellulose forculturing BLAST cell populations is an amount that enables the BLAST-NEMcells to associate as groups (i.e. clumps or clusters) of cells, therebystimulating growth and/or differentiation of the BLAST cells intoBLAST-NEM cells. A preferred amount of methyl cellulose in which toculture a BLAST cell population of the present invention to obtain aBLAST-NEM is from about 0.25% to about 2.0%, more preferably from about0.5% to about 1.5%, and even more preferably at about 1%.

A BLAST-NEM cell population of the present invention is derived byculturing a population of BLAST cells for a suitable amount of time toproduce a BLAST-NEM cell population able to develop into a hematopoieticlineage. In particular, the present invention includes a population ofBLAST-NEM cells that are derived by culturing a population of BLASTcells for a suitable amount of time to produce a population of BLAST-NEMcells that are capable of developing into erythrocyte or leukocytecells. A BLAST-NEM cell population is derived by culturing a populationof BLAST cells from about 2 days to about 12 days. A preferred BLAST-NEMcell population is derived by culturing a population of BLAST cells fromabout 4 days to about 8 days, with culturing for about 6 days being morepreferred.

In accordance with the present invention, other culture conditions (i.e.in addition to time and medium) are also important in obtaining aBLAST-NEM cell population of the present invention from a population ofBLAST cells. During culturing, variables such as cell density,temperature and CO₂ levels can be controlled to maximize the developmentof populations of BLAST-NEM cells. For example, it appears that thedensity of cells in a BLAST cell culture can affect the development of aBLAST-NEM cell population. The optimum cell density for the growth of anBLAST-NEM cell population is from about 5×10⁴ BLAST cells per ml toabout 7.5×10⁵ BLAST cells per ml, more preferably from about 1×10⁵ BLASTcells per ml to about 6×10⁵ BLAST cells per ml, and even more preferablyfrom about 2×10⁵ BLAST cells per ml to about 5×10⁵ BLAST cells per ml.The optimum temperature for the development of an BLAST-NEM cellpopulation is from about 35° C. to about 39° C., preferably from about36° C. to 38° C., with a temperature of 37° C. being even morepreferred. The optimum CO₂ levels in the culturing environment for thedevelopment of BLAST-NEM cell populations is from about 3% CO₂ to about10% CO₂, more preferably from about 4% CO₂ to about 6% CO₂, and evenmore preferably about 5% CO₂.

In a preferred embodiment, a BLAST-NEM cell population of the presentinvention is derived by culturing a population of BLAST cells in anembryoid body medium including IMDM, with about 10% PP-FBS, 1% methylcellulose, and a mixture of growth factors including C-kit ligand, IL-1,IL-3, IL-6, IL-11, VEGF and EPO for about 6 days at about 37° C., in anabout 5% CO₂ -containing environment to obtain a population of BLAST-NEMcells.

Another aspect of the present invention is a method to produce apopulation of leukocytes and erythrocytes that includes the steps of:(a) culturing a BLAST cell population in an BLAST-NEM cell mediumincluding one or more BLAST-NEM cell growth factors to produce aBLAST-NEM cell population; and (b) culturing the BLAST-NEM cellpopulation with one or more leukocyte and/or erythrocyte growth factorsto obtain a mixed population of leukocyte and/or erythrocyte cells.Preferred leukocyte and/or erythrocyte growth factors useful for theproduction of a population of leukocytes and erythrocytes include one ormore of the leukocyte and/or erythrocyte growth factors C-kit ligand,IL-1, IL-3, IL-6, IL-11, EPO, GM-CSF, G-CSF, M-CSF, homologues of suchgrowth factors, or mixtures of such growth factors and/or homologues.More preferred leukocyte and/or erythrocyte growth factors include C-kitligand, IL-1, IL-3, IL-6, IL-11, EPO, GM-CSF, G-CSF and/or M-CSF.

A mixed population of leukocyte and/or erythrocyte cells is derived byculturing a population of BLAST-NEM cells from about 2 days to about 14days. A preferred mixed population of leukocyte and/or erythrocyte cellsis derived by culturing a population of BLAST-NEM cells from about 4days to about 12 days, with culturing for about 8 days being morepreferred.

According to the present invention, a BLAST-NEM cell medium of thepresent invention includes PP-FBS or pre-selected normal FCS as well asone or more suitable growth factor as described above. A preferredBLAST-NEM cell medium of the present invention includes from about 5% toabout 30%, more preferably from about 7% to about 20%, and even morepreferably about 10% PP-FBS or pre-selected normal FCS.

Also according to the present invention, a BLAST cell population of thepresent invention is cultured in methyl cellulose to obtain a populationof BLAST-NEM cells. A suitable amount of methyl cellulose for culturingBLAST cell populations is an amount that enables the BLAST-NEM cells toassociate as groups (i.e. clumps or clusters) of cells, therebystimulating growth and/or differentiation of the BLAST cells intoBLAST-NEM cells. A preferred amount of methyl cellulose in which toculture a BLAST cell population of the present invention to obtain aBLAST-NEM is from about 0.25% to about 2.0%, more preferably from about0.5% to about 1.5%, and even more preferably at about 1%.

A BLAST-NEM cell population of the present invention is derived byculturing a population of BLAST cells for a suitable amount of time toproduce a BLAST-NEM cell population able to develop into a hematopoieticlineage. In particular, the present invention includes a population ofBLAST-NEM cells that are derived by culturing a population of BLASTcells for a suitable amount of time to produce a population of BLAST-NEMcells that are capable of developing into a primitive erythroid cell, adefinitive erythroid cell, a macrophage, a neutrophil or a mast cell,when cultured under appropriate conditions. A BLAST-NEM cell populationis derived by culturing a population of BLAST cells from about 3 day toabout 10 days, preferably for about 6 days.

In accordance with the present invention, other culture conditions (i.e.in addition to time and medium) are also important in obtaining aBLAST-NEM cell population of the present invention from a population ofBLAST cells. During culturing, variables such as cell density,temperature and CO₂ levels can be controlled to maximize the developmentof populations of BLAST-NEM cells. For example, it appears that thedensity of cells in a BLAST cell culture can affect the development of aBLAST-NEM cell population. The optimum cell density for the growth of anBLAST-NEM cell population is from about 5×10⁴ BLAST cells per ml toabout 7.5×10⁵ BLAST cells per ml, more preferably from about 1×10⁵ BLASTcells per ml to about 6×10⁵ BLAST cells per ml, and even more preferablyfrom about 2.5×10⁵ BLAST cells per ml to about 5×10⁵ BLAST cells per ml.The optimum temperature for the development of an BLAST-NEM cellpopulation is from about 35° C. to about 39° C., preferably from about36° C. to 38° C., with a temperature of 37° C. being even morepreferred. The optimum CO₂ levels in the culturing environment for thedevelopment of BLAST-NEM cell populations is from about 3% CO₂ to about10% CO₂, more preferably from about 4% CO₂ to about 6% CO₂, and evenmore preferably about 5% CO₂.

In a preferred embodiment, a BLAST-NEM cell population of the presentinvention is derived by culturing an individual BLAST cell colony in amedium including IMDM, with about 10% PP-FBS, 1% methyl cellulose, and amixture of growth factors including IL-1, IL-3, IL-6, IL-11, C-kitligand and EPO for about 6 days at about 37° C., in an about 5% CO₂-containing environment to obtain a population of BLAST-NEM cells.

Another aspect of the present invention is a method to produce animmortalized precursor cell population by (a) transforming an embryonicstem cell population of the present invention with an immortalizing geneto create a transformed stem cell population; (b) culturing thetransformed stem cell population under effective conditions to produce atransformed embryoid body cell population; and (c) incubating thetransformed embryoid body cell population under conditions suitable toobtain an immortalized precursor cell population.

Methods for transformation and expression of immortalizing genes in anembryonic cell population of the present invention are standard to thosein the art (see, for example, Sambrook et al., ibid.). A preferredimmortalizing gene of the present invention includes a gene that encodesa protein that is capable of altering an embryonic cell of the presentinvention in such a manner that the cell can survive under appropriateculture conditions for at least about 1 month, preferably about 6 monthsand even more preferably about 12 months. A preferred immortalizing geneof the present invention is a HOX11 gene. A more preferred immortalizinggene is a human HOX11 gene (described in detail in Lu et al., EMBO J.10:2905-2910, 1991).

Preferably, a HOX11 gene of the present invention is operatively linkedto an expression vector to from a recombinant molecule. The phrase"operatively linked" refers to insertion of a nucleic acid molecule(e.g., a gene) into an expression vector in a manner such that themolecule is able to be expressed when transformed into a host cell. Asused herein, an expression vector is a DNA or RNA vector that is capableof transforming a host cell and of effecting expression of a specifiednucleic acid molecule. Preferably, the expression vector is also capableof replicating within the host cell. Expression vectors can be eitherprokaryotic or eukaryotic, and are typically viruses or plasmids.Expression vectors of the present invention include any vectors thatfunction (i.e. direct gene expression) in embryonic cells of the presentinvention. Preferred expression vectors of the present invention candirect gene expression in mammalian cells and more preferably in thecell types heretofore disclosed. Preferred expression vectors of thepresent invention include, for example, MSCV and MESV (described in Grezet al., Proc. Natl. Acad. Sci. USA 87:9202-9206, 1990), with MSCVv2.1retroviral expression vector being particularly preferred.

A preferred recombinant molecule of the present invention comprises anMSCV-HOX11 plasmid (described in detail in Example 13).

According to the present invention, an effective condition to produce atransformed embryoid body cell population of the present inventionincludes those conditions disclosed herein that are suitable for thedevelopment of an EB cell population. Preferably, a transformedembryonic stem cell population of the present invention is cultured forabout 12 days, more preferably for about 8 days and even more preferablyfor about 7 days.

Preferred conditions suitable to produce an immortalized precursor cellpopulation of the present invention include suitable culture medium(referred to herein as IPC medium) and culture times. A preferred IPCmedium of the present invention includes at least one growth factor asdisclosed herein. A more preferred IPC medium of the present inventionincludes IL-3, IL-4, IL-6, IL-11, EPO, C-kit ligand, LIF, or mixtures ofsuch growth factors and/or homologues of such growth factors. An evenmore preferred IPC medium of the present invention includes a mixture ofIL-3 and EPO, C-kit ligand combined with EPO and/or homologues thereof.

In accordance with the present invention, an IPC medium includes PP-FBSor pre-selected normal FCS, in addition to one or more growth factorsdescribed above. A preferred concentration of PP-FBS or pre-selectednormal FCS to include in an IPC medium of the present invention includesfrom about 10% and about 25%, more preferably from about 12% to about20%, and even more preferably about 15% PP-FBS or pre-selected normalFCS.

Also according to the present invention, a transformed EB cellpopulation of the present invention is cultured in liquid culture toobtain a population of immortalized precursor cells of the presentinvention.

A population of immortalized precursor cells of the present invention isderived by culturing a population of transformed EB cells at a suitablecell density to produce a precursor population of immortalized cells.The optimum cell density for the growth of a transformed EB cellpopulation is preferably from about 1×10⁵ cells to about 7×10⁵transformed EB cells, more preferably from about 2×10⁵ cells to about6×10⁵ transformed EB cells, and even more preferably about 5×10⁵transformed EB cells per ml of culture medium.

Applicants have discovered that culturing of a transformed EB cellpopulation for a certain period of time in accordance with the presentinvention results in the formation of a precursor population ofimmortalized cells of the present invention. Preferably, a precursorpopulation of immortalized cells is derived by culturing a population oftransformed EB cells from at least about 1 days to about 28 days, morepreferably from at least about 2 days to about 14 days and even morepreferably from about 3 days to about 8 days.

Other culture conditions (i.e. in addition to time and medium) which caneffect the development of a precursor population of immortalized cellsof the present invention include the temperature and CO₂ content of theculture environment as disclosed in detail herein.

In a preferred embodiment, a precursor population of immortalized cellsof the present invention is derived by culturing a population oftransformed EB cells of the present invention in an IPC medium includingIMDM, with about 10% pre-selected normal FCS, 1% methyl cellulose, andeither a mixture of growth factors including IL-3 and EPO or C-kitligand and EPO. The transformed EB cell population is grown at a celldensity of from about 2×10⁵ cells per ml of medium to about 5×10⁵ cellsper ml of medium. After reaching that density, the transformed EB cellpopulation is then cultured for from about 70 to about 100 days, atabout 37° C., in an about 5% CO₂ -containing environment to obtain apopulation of immortalized precursor cells.

An immortalized precursor cell population of the present inventionincludes cells of mesodermal cell lineage. In particular, animmortalized precursor cell population of the present invention includescells of hematopoietic lineage, endothelial lineage, epithelial lineage,muscle cell lineage and neural cell lineage. A preferred immortalizedprecursor cell population of the present invention includes cellscapable of developing into erythroid cells, endothelial cell andleukocyte lineage, and progenitors and progeny thereof.

According to the present invention, a precursor cell population of thepresent invention is immortalized using a HOX11 gene. Such immortalizedcell populations are referred to herein as HOX11 precursor cellpopulations. In one embodiment, HOX11 precursor cell populations of thepresent invention include: (1) a population of cells comprising cellshaving a cell surface molecule FcγRII, FcγRIII, Thy-1, CD44, VLA-4α,LFA-1β or combinations thereof; (2) a population of cells comprisingcells having a cell surface molecule FcγRII, FcγRIII, CD44, VLA-4α,LFA-1β or combinations thereof; (3) a population of cells comprisingcells having a cell surface molecule HSA, (heat stable antigen, alsoreferred to herein as CD24) CD44, VLA-4α, LFA-1β, ICAM-1 or combinationsthereof; (4) a population of cells comprising cells having a cellsurface molecule CD45, Aa4.1, Sca-1, HSA, FcγRII, FcγRIII, Thy-1, Mac-1,Gr-1, CD44, VLA-4α, LFA-1β or combinations thereof; or (5) a populationof cells comprising cells having a cell surface molecule CD45, Aa4.1,HSA, FcγRII, FcγRIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α, LFA-1β, ICAM-1or combinations thereof. According to the present invention, the cellsof group (1), (4) and (5) can either express or not express the cellsurface marker TER119. In addition, the cells of group (2) can eitherexpress or not express the cell surface marker Thy-1.

In another embodiment, a HOX11 precursor cell population of the presentinvention comprises: (1) a population of cells that is responsive to agrowth factor selected from the group consisting of IL-3, IL-4, IL-6,IL-11, EPO, C-kit ligand, LIF or mixtures thereof; (2) a population ofcells that is responsive to a growth factor selected from the groupconsisting of IL-3, EPO, C-kit ligand, LIF or mixtures thereof; or (3) apopulation of cells that is responsive to a growth factor selected fromthe group consisting of IL-3, EPO, granulocyte macrophage colonystimulating factor (GM-CSF) or mixtures thereof.

Particularly preferred HOX11 precursor cell populations of the presentinvention comprise: (1) a population of cells that comprise a cellsurface molecule selected from the group consisting of FcγRII, FcγRIII,Thy-1, CD44, VLA-4α, LFA-1β or combinations thereof, and are responsiveto a growth factor selected from the group consisting of IL-3, IL-4,IL-6, IL-11, EPO, C-kit ligand, LIF or mixtures thereof; (2) apopulation of cells that comprise a cell surface molecule selected fromthe group consisting of HSA, CD44, VLA-4α, LFA-1β, ICAM-1 orcombinations thereof, and are responsive to a growth factor selectedfrom the group consisting of IL-3, EPO, C-kit ligand, LIF or mixturesthereof; and (3) a population of cells that comprise a cell surfacemolecule selected from the group consisting of CD45, Aa4.1, Sca-1, HSA,FcγRII, FcγRIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α, LFA-1β orcombinations thereof, and are responsive to a growth factor selectedfrom the group consisting of IL-3, EPO, GM-CSF or mixtures thereof.

In another embodiment, a HOX11 precursor cell population of the presentinvention comprises a population of cells that express RNA transcribedfrom the βH1, zeta (ζ) and β major globin genes.

In a preferred embodiment, a precursor cell population of the presentinvention comprises: (1) a population of cells, referred to herein asEmbryoid Body HOX11 Cell Line-1 (EBHX-1), that comprises a cell surfacemolecule selected from the group consisting of FcγRII, FcγRIII, Thy-1,CD44, VLA-4α, LFA-1β or combinations thereof, that is responsive to agrowth factor selected from the group consisting of IL-3, IL-4, IL-6,IL-11, EPO, C-kit ligand, LIF or mixtures thereof, and that expressesRNA transcribed from the βH1, ζ and β major globin genes; (2) apopulation of cells, referred to herein as Embryoid Body HOX11 CellLine-4 (EBHX-4), that comprises a cell surface molecule selected fromthe group consisting of FcγRII, FcγRIII, CD44, VLA-4α, LFA-1β orcombinations thereof; (3) a population of cells, referred to herein asEmbryoid Body HOX11 Cell Line-11 (EBHX-11), that comprises a cellsurface molecule selected from the group consisting of HSA, CD44,VLA-4α, LFA-1β, ICAM-1 or combinations thereof, that is responsive to agrowth factor selected from the group consisting of IL-3, EPO, C-kitligand, LIF or mixtures thereof, and that expresses RNA transcribed fromthe βH1, ζ and β major globin genes; (4) a population of cells, referredto herein as Embryoid Body HOX11 Cell Line-14 (EBHX-14), that comprisesa cell surface molecule selected from the group consisting of CD45,Aa4.1, Sca-1, HSA, FcγRII, FcγRIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α,LFA-1β or combinations thereof, that is responsive to a growth factorselected from the group consisting of IL-3, EPO, GM-CSF or mixturesthereof; and (5) a population of cells, referred to herein as EmbryoidBody HOX11 Cell Line-15 (EBHX-15), that comprises a cell surfacemolecule selected from the group consisting of CD45, Aa4.1, HSA, FcγRII,FcγRIII, Thy-1, Mac-1, Gr-1, CD44, VLA-4α, LFA-1β, ICAM-1 orcombinations thereof.

In another preferred embodiment, a precursor cell population of thepresent invention comprises populations of cells referred to herein asEmbryoid Body HOX11 Cell Line-2 (EBHX-2), Embryoid Body HOX11 CellLine-3 (EBHX-3), Embryoid Body HOX11 Cell Line-5 (EBHX-5), Embryoid BodyHOX11 Cell Line-6 (EBHX-6), Embryoid Body HOX11 Cell Line-7 (EBHX-7),Embryoid Body HOX11 Cell Line-8 (EBHX-8), Embryoid Body HOX11 CellLine-9 (EBHX-9), Embryoid Body HOX11 Cell Line-10 (EBHX-10), EmbryoidBody HOX11 Cell Line-12 (EBHX-12), and Embryoid Body HOX11 Cell Line-13(EBHX-13), the production of which are described in Example 14.

According to the present invention, a population of immortalizedprecursor cells is preferably at least about 70% clonal, more preferablyat least about 80% clonal and even more preferably at least about 90%clonal. As used herein, the term "clonal" refers to a group of cellsthat are of a single cell type (e.g., that all express the same surfacemarkers or display essentially the same responsiveness to a growthfactor).

The pluripotent and/or precursor cell populations of the presentinvention can be used in the isolation and evaluation of compoundsassociated with the differentiation of embryonic cells. Thus, anotheraspect of the present invention is a method to identify a compoundexpressed during the development of a population of embryonic stem cellswhich, as used herein, is a compound expressed during the development ofa population of BLAST cells of the present invention from an ES cellpopulation (i.e. including the stage of EB cell development) intransformed or non-transformed cells. The method comprisescharacterizing at least a portion of the cellular composition of atleast one cell contained in a population of cells including an ES cellpopulation, a pluripotent EB cell population of the present invention, aHOX11 precursor cell population, a pluripotent embryonic blast cellpopulation and intermediates thereof (i.e. cells of stages between ESand EB cell populations, or between EB and BLAST cell populations), toidentify a compound expressed during the development of a population ofembryonic stem cells. As used herein, a cellular composition refers acomposition containing components of a cell. Preferred cellularcompositions of the present invention include nucleic acids, proteins,lipids (including membranes) and/or carbohydrates, with proteins, DNAmolecules and RNA molecules being more preferred. Preferred cells fromwhich to identify compounds includes, for example, EBHX-1, EBHX-2,EBHX-3, EBHX-4, EBHX-5, EBHX-6, EBHX-7, EBHX-8, EBHX-9, EBHX-10,EBHX-11, EBHX-12, EBHX-13, EBHX-14 and EBHX-15 (the group of which arereferred to herein as EBHX-1 through EBHX-15).

The present invention includes a variety of methods to identify anembryonic cell compound using an embryonic cell population of thepresent invention. In one embodiment, an embryonic cell compound of thepresent invention is identified by direct hybridization studies,comprising hybridizing a nucleic acid molecule probe (which can be DNA,RNA or modified forms thereof) to a composition of nucleic acidmolecules isolated from an embryonic cell population of the presentinvention. Such a method is useful for identifying the expression ofcompounds in an embryonic cell population. For example, a nucleic acidmolecule encoding a protein can be hybridized under suitable conditionsknown to those of skill on the art (see, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press,1989) to an RNA composition isolated from an embryonic cell populationof the present invention, or to a cDNA product thereof. Preferrednucleic acid molecules for use in a direct hybridization study of thepresent invention include nucleic acid molecules that encode markerproteins including, but not limited to, endothelial cell proteins,lymphoid cell proteins, epithelial proteins, mature hematopoietic cellproteins, and/or hematopoietic stem cell proteins. As used herein, amarker protein is a protein typically found in certain cell types and,as such, can suggest identification of such cell type. Particularlypreferred nucleic acid molecules for use in a direct hybridization studyof the present invention include nucleic acid molecules that encodeproteins including stem cell leukemia protein, GATA-1, GATA-2, C-Myb,C-kit ligand, C-fms, Flk-1, X protein (encoded by a ζ globin gene), βmajor globin protein, ζ protein (encoded by a βH1-globin gene), Yprotein (encoded by an εY gene), brachyury, VLA-4 and LFA-1. Anembryonic cell-derived nucleic acid composition useful for such directhybridization studies can include genomic DNA, RNA or cDNA of such RNA.

In another embodiment, an embryonic cell compound of the presentinvention is identified by selective nucleic acid hybridizationtechniques well known to those of skill in the art. Such subtractivehybridization techniques are particularly useful for identifying novelembryonic cell compounds and for identifying compounds expressed in agiven cell type. Subtractive hybridization techniques of the presentinvention can be performed by, for example: (1) hybridizing nucleic acidmolecules isolated or derived from an embryonic cell population of thepresent invention to nucleic acid molecules isolated or derived from anon-embryonic cell population; or (2) hybridizing nucleic acid moleculesisolated or derived from a first embryonic cell population of thepresent invention to nucleic acid molecules isolated or derived from asecond embryonic cell population of the present invention. For example,nucleic acid molecules isolated from an EB cell population of thepresent invention can be subtracted from nucleic acid molecules isolatedor derived from a BLAST cell population of the present invention or viceversa.

In yet another embodiment, an embryonic cell compound of the presentinvention is identified by nucleotide sequencing of DNA isolated from anembryonic cell population of the present invention. In order to identifycompounds expressed in certain cell types, cDNA copies of poly A+ RNA ispreferably analyzed. Identification of embryonic cell compounds can beachieved by comparing the DNA sequence information encoding suchcompounds derived from the embryonic cell population with sequences ofknown molecules. Such DNA sequencing studies are particularly useful foridentifying novel embryonic cell compounds. DNA sequencing studies canbe performed using techniques standard in the art (see, for example,Sambrook et al., ibid.).

In yet another embodiment, an embryonic cell compound of the presentinvention is identified by selective binding of proteins isolated froman embryonic cell population of the present invention to antibodiesspecific for known cellular proteins to determine the presence of suchcellular proteins in the embryonic cell population. Such antibodybinding studies are particularly useful for identifying the expressionof known compounds by embryonic cell populations of the presentinvention. Antibody binding studies of the present invention can beperformed using techniques standard in the art, such as by immunoblotassays, immunoprecipitation assays, enzyme immunoassays (e.g., ELISA),radioimmunoassays, immunofluorescent antibody assays and immunoelectronmicroscopy; see, for example, Sambrook et al., ibid.

In yet another embodiment, an embryonic cell compound of the presentinvention is identified by cell culture assays that indicate cellsurvival and cell proliferation. Such cell culture assays areparticularly useful for identifying both novel and known growth factorsthat are secreted by an embryonic cell population of the presentinvention. A cell culture assay of the present invention can beperformed by: (1) recovering supernatant from a culture of a denseembryonic cell population of the present invention; (2) contacting thesupernatant with a sparse population of the embryonic cell population;and (3) determining if the supernatant is able to promote the survivaland/or proliferation of said embryonic cell population by observing thehealth of said cell population. Such cell culture assays can beperformed using the cell culturing techniques disclosed in detailherein. A preferred dense population of cells includes any cell densityused to culture an embryonic cell population as disclosed herein. Apreferred sparse population of an embryonic cell population of thepresent invention includes a cell density of from about 5×10³ to about2×10⁵ cells per ml.

In yet another embodiment, an embryonic cell compound of the presentinvention involved in signal transduction in an embryonic cell isidentified using kinase assays that are standard in the art. Such kinaseassays are particularly useful for identifying known signal transductionproteins in an embryonic cell population of the present invention.

In yet another embodiment, an embryonic cell compound of the presentinvention is identified by protein:protein binding studies other thanantibody binding studies. In particular, embryonic cell compounds areidentified by determining ligand:receptor interactions. For example, anembryonic cell population of the present invention can be contacted witha known ligand to determine if the cell population contains cells havingthe receptor to which the ligand can bind. Such protein:protein bindingstudies can be performed using techniques known to those of skill in theart.

According to the present invention, an embryonic cell compound can be acompound that has been previously identified, or not previouslyidentified, from a cell or culture medium of a cell other than apopulation of cells of the present invention. For example, an embryoniccell compound of the present invention can include a growth factor thatis also produced by a more mature fetal or adult cell of an animal.

An embryonic cell compound of the present invention can be a compoundthat is capable of having a biological effect on a cell. For example,preferred embryonic cell compounds are capable of maintaining thesurvival of a cell, inducing the propagation of a cell and/orstimulating the differentiation of a cell. Preferred embryonic compoundsof the present invention include a compound that can be used as a markerfor a population of embryonic cells. In particular, an embryonic cellmarker of the present invention can be cell surface markers, secretedmolecules, cytoplasmic signal transduction molecules, transcriptionfactors and other DNA or RNA binding proteins. As used herein, a cellsurface marker refers to any compound on the surface of a cell that isdetectable by techniques such as antibody binding studies, gelelectrophoresis and various chromatography techniques known to those ofskill in the art. A cell surface marker can include cell surfacereceptors, adhesion proteins, cell surface carbohydrate moieties,membrane-bound ligands and other molecules involved in cell to cellcommunication. A secreted molecule refers to any molecule produced andsecreted by a cell into an extracellular environment and includes growthfactors and other ligands. A cytoplasmic signal transduction moleculerefers to a molecule that is able to regulate an intracellular chemicalreaction that enables a cell to modify its biological functions based onsignals in the environment, either outside or inside the cell. Signaltransduction molecules can include enzymes, such as kinases,phosphatases and phospholipases. Preferred embryonic cell compounds ofthe present invention include a cell surface receptor, a cell surfacemolecule, a cytoplasmic signal transduction protein, a transcriptionfactor, a growth factor, and DNA or RNA binding proteins.

Identification of known and novel (i.e. newly identified) compounds inan embryonic cell population of the present invention is particularlyuseful for defining markers useful for the identification and/orisolation of comparable populations of cells from non-embryonicpopulations of cells. A particularly preferred non-embryonic cellpopulation to look for cells having embryonic markers includesnon-embryonic cell populations, including bone marrow (e.g., fetal,infant, adolescent and adult bone marrow). The presence of an embryoniccell marker of the present invention on a non-embryonic cell canindicate that the non-embryonic cell is pluripotent. Preferred embryoniccell population markers to identify comparable non-embryonic cellpopulations include lineage-specific markers, such as hematopoieticprecursor markers, in particular pre-hematopoietic mesoderm markers. Ina preferred embodiment, a population of adult bone marrow cells isscreened for the presence of a cell surface marker found on the surfaceof an EB cell population and/or a BLAST cell population of the presentinvention.

One embodiment of the present invention is a formulation that containsone or more isolated embryonic cell compounds of the present inventionthat can be used for therapeutic or experimental use. According to thepresent invention, an isolated embryonic cell compound is a compoundthat has been removed from its natural milieu. An isolated embryoniccell compound can, for example, be obtained from its natural source, beproduced using recombinant DNA technology, or be synthesized chemically.Preferred embryonic cell compounds of the present invention, includinghomologues thereof, are capable of regulating embryonic development. Apreferred embryonic cell compound homologue includes at least oneepitope capable of effecting differentiation of an ES cell population.The ability of an embryonic cell compound homologue to effectdifferentiation of an ES cell population can be tested using techniquesdisclosed herein. A preferred formulation of the present inventionincludes at least one protein secreted by a cell contained in an EB cellpopulation of the present invention and/or a BLAST cell population ofthe present invention. Preferably, a formulation of the presentinvention comprises a culture supernatant obtained by culturing an EBcell and/or BLAST cell population of the present invention.

Another aspect of the present invention comprises an antibody capable ofbinding to a cell compound of a cell population of the presentinvention. Binding can be measured using a variety of methods known tothose skilled in the art including immunoblot assays,immunoprecipitation assays, enzyme immunoassays (e.g., ELISA),radioimmunoassays, immunofluorescent antibody assays and immunoelectronmicroscopy; see, for example, Sambrook et al., ibid. Antibodies of thepresent invention can be either polyclonal or monoclonal antibodies.Antibodies of the present invention include functional equivalents suchas antibody fragments and genetically-engineered antibodies, includingsingle chain antibodies, that are capable of selectively binding to atleast one of the epitopes of the protein or mimetope used to obtain theantibodies. Preferred antibodies are raised in response to surfacemarker proteins of an embryonic cell population of the presentinvention, in particular, surface cell receptors. Antibodies of thepresent invention can be produced using methods standard in the art.Antibodies of the present invention are particularly useful foridentifying and isolating populations of cells having such surfacemarkers, in particular, populations of embryonic cells from differentspecies of animals and/or cells with similar markers from adult bonemarrow. Thus, particularly preferred antibodies of the present inventioninclude antibodies that are capable of binding to cellular markers thatdelineate between different embryonic cell populations of the presentinvention.

One embodiment of the present invention includes a method to produce anantibody, comprising administering to an animal an effective amount of aprotein derived from a HOX11 precursor cell population of the presentinvention and recovering an antibody capable of selectively binding tosuch protein. According to the present invention, a protein derived froma HOX11 precursor cell population can include an isolated protein or aprotein that is associated with a HOX11 precursor cell population. Anisolated protein useful for producing antibodies can include the fulllength protein, as well as fragments thereof, in particular, peptides.Such fragments can be selected by those of skill in the art based uponthe antigenicity (i.e. ability to induce an antibody response in ananimal) of a particular fragment. The antigenicity of a fragment can bedetermined by trial and error without experimentation, or by computeranalysis using standard programs.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of a proteinisolated from a HOX11 precursor cell population of the present inventionto produce the antibodies and (b) recovering the antibodies. Anotherpreferred method to produce antibodies of the present invention includes(a) administering to an animal an effective number of cells from a HOX11precursor cell population of the present invention to produce theantibodies and (b) recovering the antibodies. A preferred cellpopulation for use with the present method includes EBHX-1 throughEBHX-15, and/or derivatives thereof.

Another aspect of the present invention is a therapeutic compositionthat comprises a cell population of the present invention, which iscapable of serving as a population of cells that act as progenitors ofvarious lineages. The therapeutic composition can be particularly usefulto repopulate one or more lineages in an animal. As used herein, theterm repopulate refers to a cell population that can be administered toan animal to restore a lineage of cells. A therapeutic composition ofthe present invention can be useful for the treatment of disease, suchas anemia, leukemia, breast cancer and other solid tumors, and AIDS. Atherapeutic composition of the present invention can be particularlyuseful for enhancing populations of adult bone marrow cells used intransplantation procedures. A preferred therapeutic composition of thepresent invention includes a population of EB cells of the presentinvention, a population of immortalized precursor cells of the presentinvention and/or a population of BLAST cells of the present invention. Amore preferred therapeutic composition of the present invention includesa population of EB cells of the present invention, a population ofimmortalized precursor cells of the present invention or derivatives(i.e., any cell that is derived from an immortalized precursor cellpopulation) thereof and/or a population of BLAST cells of the presentinvention derived from an ES cell population derived from a mammalianembryo. An even more preferred therapeutic composition of the presentinvention includes a population of EB cells of the present invention, apopulation of BLAST cells of the present invention derived from an EScell population derived from a human embryo, and/or EBHX-1 throughEBHX-15 and/or derivatives thereof.

In one embodiment, the present invention includes a method to repopulatea hematopoietic cell population in an animal, comprising administeringto an animal a suitable number of cells of a HOX11 pluripotent cellpopulation of the present invention. A suitable number of cells includesa number needed to, for example, repopulate a lymphocyte population in asubjected being treated for a lymphoma. For example, subjects having alymphoma can receive large doses of chemotherapy and radiotherapy todestroy cancerous lymphocyte cells. The lymphocyte populations in thesesubjects can be repopulated by administering to the subject a suitablenumber of cells from a cell population of the present invention, suchthat the lymphocyte count of the subject returns substantially back tonormal. Preferably, a HOX11 pluripotent cell population for use with thepresent method includes EBHX-1, EBHX-4, EBHX-11, EBHX-14 and/or EBHX-15cells or derivatives thereof.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals, and more preferably to humans.Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate and thatmaintains the integrity of the embryonic cell population. Examples ofsuch excipients include aqueous physiologically balanced salt solutions.Excipients can also contain minor amounts of additives, such assubstances that enhance isotonicity and chemical stability.

One embodiment of the present invention includes a method to identify aregulatory factor that influences the growth of a cell, comprising: (1)contacting a HOX11 precursor cell population of the present inventionwith a regulatory factor including a putative regulatory factor, a knownregulatory factor and mixtures thereof; and (2) assessing theresponsiveness of the precursor cell population to the regulatoryfactor. Such method is particularly useful for identifying factorsuseful as therapeutic reagents to treat hematopoietic disorders. As usedherein, the term "putative" refers to compounds having an unknown orpreviously unappreciated regulatory activity in a particular process. Aputative compound can be a protein-based compound, a carbohydrate-basedcompound, a lipid-based compound, a nucleic acid-based compound, anatural organic compound, a synthetically derived organic compound, ananti-idiotypic antibody and/or catalytic antibody, or fragments thereof.A putative regulatory compound can be obtained, for example, fromlibraries of natural or synthetic compounds, in particular from chemicalor combinatorial libraries (i.e., libraries of compounds that differ insequence or size but that have the same building blocks; see forexample, U.S. Pat. Nos. 5,010,175 and 5,266,684 of Rutter and Santi,which are incorporated herein by reference in their entirety) or byrational drug design.

In a rational drug design procedure, the three-dimensional structure ofa compound, such as a cell surface marker, can be analyzed by, forexample, nuclear magnetic resonance (NMR) or x-ray crystallography. Thisthree-dimensional structure can then be used to predict structures ofpotential compounds, such as putative regulatory compounds by, forexample, computer modelling. The predicted compound structure can thenbe produced by, for example, chemical synthesis, recombinant DNAtechnology, or by isolating a similar molecule from a natural source(e.g., plants, animals, bacteria and fungi). Potential regulatorycompounds can also be identified using SELEX technology as described in,for example, PCT Publication Nos. WO 91/19813; WO 92/02536 and WO93/03172 (which are incorporated herein by reference in their entirety).

One embodiment of a putative regulatory reagent includes a neutralizingreagent capable of blocking the activity of a protein that controls cellresponsiveness (i.e., growth and/or differentiation) to a compound, suchas a growth factor. The present invention includes a method to identifya neutralizing reagent, comprising: (1) contacting a HOX11 precursorcell population of the present invention with a known regulatory factorto produce a controlled cell population; (2) combining the controlledcell population with a neutralizing reagent including a knownneutralizing compound of the regulatory factor or a putativeneutralizing compound of the regulatory factor; and (3) assessing theresponsiveness of the precursor cell population to the neutralizingcompound.

According to the present method, the step of assessment can be performedusing any one of a variety of methods known to those of skill in theart. In particular, the assessment step can be performed using aproliferation assay and/or a differentiation assay. A preferredproliferation assay of the present invention comprises standard assaysthat determine cell count number, thymidine uptake by a cell and enzymeactivity, including enzyme-linked immunoassays and cellular enzymeassays. A preferred differentiation assay of the present inventioncomprises a standard method including: (a) determining globin geneexpression; (b) identifying cell surface markers; (c) determiningresponsiveness to a growth factor; (d) observing alterations inmorphology; and (e) determining expression of genes associated withdifferentiation of hematopoietic cells.

Another aspect of the present invention is the use of a cell populationof the present invention for the treatment of genetic diseases. Geneticdiseases associated with various lineages can be treated by geneticmodification of autologous or allogenic populations of embryonic cellsof the present invention. For example, diseases such asbeta-thalassemia, sickle cell anemia, adenosine deaminase deficiency andother genetic diseases related to a deficiency or malfunction of a cellof hematopoietic lineage, can be corrected by introduction of a wildtype gene into the embryonic cell population. Diseases other than thoseassociated with hematopoietic cells can be treated, where the disease isrelated to the lack of a particular secreted product, such as a hormone,enzyme, growth factor and the like. Specific promoters can be employedbased upon identification of transcription factors of an embryonic cellpopulation as described herein. Thus, inducible production of a desiredproduct encoded by transformed genes can be achieved. Methods fortransformation and expression of genes in an embryonic cell populationof the present invention are standard to those in the art (see, forexample, Sambrook et al., ibid.).

In accordance with the present invention, a nucleic acid molecule can betransformed into an embryonic cell population of the present inventionto inhibit particular gene products, thereby inhibiting susceptibilityto a disease. For example, an embryonic cell population of the presentinvention can be transformed with a ribozyme, or a nucleic acid moleculethat is capable of homologous recombination or antisense expression. Forexample, a BLAST cell population of the present invention can betransformed with a gene that disrupts the expression of a specific Tcell receptor gene and administered to an animal. Subsequentdifferentiation of the BLAST cell population to a T cell populationresults in the production of a population of T cell receptor negative Tcells. Such a method could be effective for preventing or treatingautoimmune disease which involve autoreactive T cell activity.Similarly, an embryonic cell population of the present invention can bemodified to introduce an antisense sequence or ribozyme to preventproliferation of any pathogen that uses proteins of an animal cell toproliferate (e.g., viruses) in an embryonic cell population, or inprogenitors or progeny thereof.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

This example describes the production of a population of embryoid bodycells from an embryonic stem cell population.

The CCE ES cell line, originally derived from a 129/Sv/Ev strain ofmouse, were maintained in Dulbecco's modified Eagles medium (DMEM)supplemented with 15% fetal calf serum (FCS), 1.5×10⁻⁴ Mmonothioglycerol (MTG), and Leukemia Inhibitory Factor (LIF). The EScells were passaged every 2-3 days at a dilution of approximately 1:15.Two days before the initiation of the differentiation cultures, theundifferentiated ES cells were passaged into Iscove's modifiedDulbecco's medium (IMDM) supplemented with the above components. Toinduce differentiation into an EB population of cells, the ES cells weretrypsinized, washed, and counted using techniques standard in the art.The freshly dissociated ES cells were then cultured in IMDM containing15% platelet-derived fetal bovine serum (PDS; obtained from Antech,Texas; also referred to herein as platelet-poor fetal bovine serum,PP-FBS), 4.5×10⁻⁴ M MTG, transferrin (300 μg), glutamine (2 mM). The EScells were plated in a final volume of 10 ml at a concentration of about3000 to about 4500 cells per ml of medium in 150 mm bacterial gradedishes. The ES cell population was then cultured in a humidifiedenvironment of 5% CO₂, at a temperature of 37° C. At an appropriate timeafter the initiation of differentiation (i.e., plating of cells inPP-FBS), EB cell populations were harvested using standard techniques.The cells were then centrifuged at ×1000 rpm to pellet the cells and thesupernatant was removed. The EB cells were resuspended in 3 ml trypsinfor 1 to 3 minutes at 37° C. An equal volume of IMDM containing 5 to 10%FCS was added to the trypsinized cells. A single cell suspension of EBcells was achieved by passing the EB cells 3 to 4 times through a 3 mlor 5 ml syringe with a 20 gauge needle.

The EB cell colonies were viewed under a Leitz inverted light microscopeand were found to generally consist of groups of tightly packed cells,in which individual cells were not easily detectable. A representativemicroscopic view of an EB cell colony is shown in FIG. 2, cell colony A.The EB cell colony consisted of a tightly packed group of cells, inwhich single cells were essentially not discernable.

Example 2

This example describes the labelling of a population of EB cells usingantibodies specific for known cell surface molecules.

EB cells derived from ES cells that had been incubated for about 4 days(Day 4 EB) according to the method described in Example 1 were labelledfor fluorescence activated cell sorter analysis using a panel ofantibodies against a number of different surface markers. These includedantibodies specific for AA4.1, Sca-1, C-kit receptor, H-2b, VLA-4, CD44,CD45 and Thy 1. In addition, a population of CCE ES cells, similar tothose cells used to derive the EB cell population, were stained underthe same conditions as the EB cell population as a control sample. Theresults are shown in FIG. 3A and 3B. Referring to FIG. 3A and 3B, theDay 4 EB cells (d4 EB) stained with low amounts of anti-Sca-1,anti-C-kit receptor and anti-H-2b antibodies, and essentially noanti-Thy 1, anti-VLA-4, anti-CD44 and anti-CD45 antibodies, therebyindicating low or no expression of the corresponding surface markerprotein. In addition, comparing the staining pattern of the EB cells tothe control ES cells, the EB cells express slightly higher levels ofC-kit receptor and H-2b protein but slightly less Sca-1 protein. Thus,the results show that expression of certain surface antigens change overa period of 4 days during development of ES cells to EB cells.

Example 3

This example describes the production of a population of embryonic blastcells from an embryoid body cell population.

EB cells were generated as described in Example 1. A series of cultureswere prepared by plating about 2×10⁵ to about 5×10⁵ EB cells, derivedaccording to the method described in Example 1. The EB cells werecultured in 1% methyl cellulose made in IMDM containing 10% PP-FBS,transferrin (300 μg/ml), glutamine (2 mM), and either a mixture of IL-1(1000 units/ml), IL-6 (5 ng/ml), IL-11 (25 ng/ml), C-kit ligand (100ng/ml), or C-kit ligand (100 ng/ml), VEGF (5 ng/ml) and EPO (2units/ml), or C-kit ligand (100 ng/ml) alone. The EB cells were culturedin a final volume of 1 ml in a 35 mm bacterial grade dishes in ahumidified environment of 5% CO₂ at 37° C. Individual EB cell cultureswere incubated for 3 days, 3.5 days, 4 days and 4.5 days. BLAST cellcolonies identified using a Leitz inverted light microscope based onmorphology. A representative BLAST cell colony is shown in FIG. 2, cellcolony B. The BLAST cell colony consisted of a clumped, but not tightlypacked, group of cells in which individual cells could be discerned inthe colony.

The formation of BLAST cell colonies at each time point was scored byeye and the kinetics of BLAST cell development are shown in FIG. 5. Theresults indicate that BLAST cell (Blast) development was detected within3 days of initiation of EB cell (EBs) incubation under the above cultureconditions, increasing slightly in number by day 3.5 and then decreasingto low levels by day 4.5. As the number of BLAST cells increased, thenumber of EB cells decreased. In addition, as the number of BLAST cellsdecreased, the number of erythroid cells (Ery), which were scored by redcolor, appeared at day 4 and continued to increase through day 4.5.

Example 4

This example describes the labelling of a population of BLAST cellsusing antibodies specific for known cell surface molecules.

EB cells were generated as described in Example 1. BLAST cells weregenerated from EB cells that had been incubated for about 6 days (Day 6BLAST) according to the method described in Example 3. The BLAST cellswere labelled for fluorescence activated cell sorter analysis using apanel of antibodies against a number of different surface markers. Theseincluded antibodies specific for AA4.1, Sca-1, C-kit receptor, H-2b,VLA-4, CD44, CD45 and Thy 1. ES cells and Day 4 EB cells (described inExample 2) were also stained as control samples. The results are shownin FIG. 3. Referring to FIG. 3, the Day 6 BLAST cells (d6 Blasts)stained with anti-CD44, anti-C-kit receptor, anti-Sca-1 and anti-VLA-4antibodies, but not with anti-H-2b, anti-Thy 1 or anti-CD45 antibodies.Thus, indicating that Day 6 BLASTS express substantial amounts of CD44,C-kit receptor, Sca-1 and VLA-4 protein, and essentially no Class IH-2^(b), Thy 1 and CD45 protein. Comparing the staining pattern of theDay 6 BLASTS with the EB cell and ES cells, the results show thatexpression of C-kit receptor, H-2b, VLA-4 and CD44 increases during the6 days of development of EB cells to BLAST cells.

Example 5

This example describes a myeloid assay to test for hematopoieticprecursors in BLAST cell populations.

A schematic representation of the myeloid assay used to identifysecondary hematopoietic colony formation from BLAST cells is shown inFIG. 6. Two different culture experiments were prepared as follows. In afirst experiment, EB cells were generated as described in Example 1 byculturing ES cells for 4 days. Individual BLAST cell colonies weregenerated by incubating the EB cell population for 3, 4, 5 and 6 daysaccording to the method described in Example 3. In a second experiment,EB cells were generated as described in Example 1 by culturing ES cellsfor 3, 3.5, 4 and 4.5 days. Individual BLAST cell colonies weregenerated by incubating each EB cell population for 6 days according tothe method described in Example 3. In each experiment, individual BLASTcell colonies were picked, dispersed in 100 μl IMDM containing 5% FCS,and transferred to a 1% methyl cellulose culture containing IMDM, 10%PP-FBS, 300 μg transferrin and a cocktail of growth factors includingIL-1 (1000 units/ml), IL-3 (100 units/ml), IL-6 (5 ng/ml), IL-11 (25ng/ml), C-kit ligand (100 ng/ml) and EPO (2 units/ml). The cultures werethen incubated for varying amounts of time in a humidified 5% CO₂, at atemperature of 37° C.

In the first experiment, developing hematopoietic colonies were scored 7days after initiation of the culture. In the second experiment,developing hematopoietic colonies were scored 3, 4, 5 and 6 days afterinitiation of the culture. The growth of hematopoietic colonies werescored based on colony morphology when the colonies were viewed under aninverted Leitz light microscope, and based on cellular stainingpatterns. Erythrocytes, macrophages, neutrophils and mast cells wereidentified in all cultures having hematopoietic cell development.

The results from the first experiment indicated that as many as 70%BLAST cells gave rise to hematopoietic colonies (see FIG. 7A). Kineticanalysis revealed that BLAST cell colonies incubated for 3 days gaverise to fewer hematopoietic colonies than BLAST cell colonies incubatedfor 6 days (see FIG. 7B). Thus, indicating that a maturation processoccurred within the BLAST cell population between days 3 and 6.

In the second experiment, the percent of total BLAST cell colonies thatgenerated a secondary hematopoietic colony, including multi- oruni-lineage colonies at each time point was determined. Multi-lineagerefers to colonies that contain erythroid plus two other lineages. Inaddition, the percent of total BLAST cell colonies that generated thatgenerated only multi-lineage colonies at each time point was determined.The results are shown in FIG. 8. Referring to the percent of cells thatreplate to multi-lineages (black bars), the results indicate that BLASTcell colonies generated from day 3 EB cells contain more immaturehematopoietic cells than those derived from day 4.5 EB cells based onthe generation of multi-lineage colonies.

Morphological analysis of the secondary hematopoietic populationsarising from BLAST cells indicated the presence of primitive erythroid(Ery^(p)), definitive erythroid (Ery^(d)) and multiple myeloid cells ina single replated culture. The results are summarized in Table 1.

                  TABLE 1    ______________________________________    Incidence of Primitive and Definitive Erythroid Colonies from    Individually Replated Blast Colonies                         Ery.sup.d Ery.sup.p + Ery.sup.d    Colony Type               Ery.sup.p (± myeloid)                                   (± myeloid)    ______________________________________    Blast Colonies               8         140       10    ______________________________________

The presence of both primitive and definitive erythroid cells in a BLASTcell colony indicates that the BLAST cell population that gave rise tothe erythroid cell populations had the potential to generate allhematopoietic populations and represents one of the earliesthematopoietic cells to develop, equivalent to a pre-yolk sac cell (seeFIG. 9, cell A). A certain number of BLAST cells generated predominantlyprimitive erythroid cells, while others generated definitive erythroidcells and cells of the various myeloid lineages. These latter twopatterns of replating indicate that some of the BLAST colonies arecommitted to primitive erythropoiesis (analogous to the cells thatexpand in the yolk sac) (FIG. 9, cell B); while others have lost thecapacity to generate this early lineage, but can generate all otherpopulations (equivalent to the cells that ultimately seed the fetalliver) (FIG. 9, cell C).

Example 6

This example describes the influence of individual growth factors onhematopoietic colony formation from BLAST cell colonies.

BLAST cell populations were generated from Day 3.5 EB cell populationsaccording to the method described in Example 3 were then incubated inthe presence of specific growth factors (shown in FIG. 10). A controlsample was prepared that contained no factors (-Factor). Twenty-fiveindividual BLAST cell colonies were picked and replated into a myeloidassay as described in Example 5. The results shown in FIG. 10A indicatethat EPO was the least effective growth factor in stimulating theformation of BLAST cell colonies capable of developing into any type ofsecondary hematopoietic colony. The results shown in FIG. 10B indicatethat IL-6 and EPO were the least effective growth factors in stimulatingthe formation of BLAST cell colonies capable of developing intomulti-lineage secondary hematopoietic colonies. C-kit ligand and amixture of C-kit ligand, EPO and VEGF were most effective in stimulatingthe formation of BLAST cell colonies capable of developing into any typeof hematopoietic colony including colonies having multi-lineagecolonies.

Example 7

This example describes the formation of a T cell population from an EScell population using a mixture of IL-7, IGF-1 and C-kit ligand growthfactors.

EB cells were generated from ES cells according to the method describedin Example 1. BLAST cell colonies were generated from the EB cellsaccording to the method described in Example 3. Individual BLAST cellcolonies were picked, dispersed in IMDM containing 5% FCS, andtransferred to a 1% methyl cellulose culture containing IMDM, 10%PP-FBS, 300 μg transferrin and a cocktail of growth factors includingIL-7, IGF-1 and C-kit ligand. The cultures were then incubated for about6 days in a humidified 5% CO₂, at a temperature of 37° C.

Thymi were obtained from pregnant (15 days gestation) outbred SwissWebster mice (purchased from Taconic) which were found to express theThy 1.1 allele. The thymi were then irradiated at a dose of 3000Gy todeplete endogenous cells. Pools of 20 BLAST cell colonies were seededinto each thymic lobe in a hanging drop culture in a terrasacki well andincubated for 48 hours. Following the 48 hour hanging-drop culture, thethymi were harvested into IMDM with 10% FCS and transferred to sterile45 micron filters (Gelman) which were placed on gelfoam sponges (Upjohn)for 3 weeks at the air medium interface. The thymi were then removedfrom the filter and dissociated by treatment with 0.25% collagenase(Sigma, St. Louis, Mo.), 10 μg/ml DNAse in phosphate buffered saline(PBS) and digested for 1 hour at 37° C. Following digestion, the thymiwere dispersed by passaging the cells through a 3 ml syringe attached toa 20 gauge needle.

The resulting single cell suspension was stained for host Thy 1.1, donorThy 1.2 and T cell receptor expression by the following method. About4×10⁴ cells were separately incubated with fluorescein isothiocyanate(FITC) labelled anti-Thy 1.2 antibody (1:1000; Pharminogen, San Diego,Calif.), FITC labelled anti-Thy 1.1 antibody (1:1000; Pharminogen),phycoerythrin (PE) labelled anti-αβ T cell receptor antibody (1:100;Pharminogen), and PE labelled anti-γδ T cell receptor antibody (1:100;Pharminogen) for 20 minutes, on ice. The cells were washed and analyzedon a FACSCAN (Becton Dickinson).

Referring to FIG. 11, donor Thy 1.2 positive cells expressing both αβand γδ T cell receptor were detected indicating that a mature T cellpopulation was derived from the donor BLAST cell colonies that had beentreated with IL-7, IGF-1 and C-kit ligand.

Example 8

This example describes the formation of a T cell population from an EScell population using varying combinations of growth factors.

BLAST cell colonies were generated as described in Example 7. IndividualBLAST cell colonies were then incubated with different combinations ofgrowth factors including C-kit ligand alone, a mixture of C-kit ligandand IL-7, a mixture of C-kit ligand, IL-7 and IGF-1 and a mixture ofC-kit ligand, VEGF and EPO. Using the method described in Example 7, theBLAST cell colonies were incubated, T cell populations were derived, andthe resulting T cell populations were stained with anti-Thy 1.2 and αβ Tcell receptor antibodies.

Referring to FIG. 12, incubation of BLAST cell colonies with thecombination of C-kit ligand, IL-7 and IGF-1 growth factors (BLASTKL/IGF-1/IL-7) produced the most αβ T cell receptor positive T cells.The presence of the other combinations of factors, however, alsoproduced some αβ T cell receptor positive T cells. A positive controlsample of bone marrow stromal cells BLAST cell colonies (BM) producedsimilar numbers of αβ T cell receptor positive T cells as theKL/IGF-1/IL-7 treated BLAST cell colonies. Thus, optimal conditions forproduction of T cell populations include incubation of BLAST cellcolonies with a mixture of C-kit ligand, IL-7 and IGF-1 growth factors.

Example 9

This example describes the formation of a B cell population from an EScell population.

EB cell colonies were generated from ES cells using the method ofExample 1. BLAST cell colonies were derived from EB colonies using themethod of Example 3. Individual BLAST cell colonies were picked frommethyl cellulose culture and transferred to confluent monolayers of S17bone marrow stromal cells that had been irradiated at a dose of 3000Gy.Cultures were grown for 4 weeks in IMDM with 5% FCS and C-kit ligand.cDNA samples were prepared from the C-kit ligand treated cells bydispersing the cells using trypsin, lysing the cells and preparing cDNAusing reverse transcriptase (BRL Gibco) according to methods standard inthe art. cDNA samples were then amplified by PCR for VDJ immunoglobulinrearrangement using a 5' VH7183 primer (5'-TGGTGGAGTCTGGGGGAGGCTTA-3';SEQ ID NO:1) and a 3' JH4 primer (5'-GGCTCCCTCAGGGACAAATATCCA-3'; SEQ IDNO:2) using the following PCR profile: 94° C. 1 minute, 72° C. 2 minutesfor 29 cycles; 94° C. 1 minute, 72° C. 10 minutes for one cycle. PCRproducts were subjected to southern blotting and hybridized with a probecomplimentary to a sequence common to immunoglobulin J regions.

The results indicate that treatment of BLAST cells with C-kit ligand inaddition to exposure to bone marrow stromal cells results in theproduction of a B cell population containing cells having rearrangedimmunoglobulin VDJ genes.

Example 10

This example describes the development mixed populations of erythroidand endothelial cells from EB cell populations.

A. Mixed Endothelial and Erythroid Population Development

Approximately 2×10⁵ EB cells derived from CCE ES cells that werecultured for about 4 days according to the method described in Example 1were dissociated with trypsin and re-plated and cultured in 1% methylcellulose made in IMDM containing 10% PP-FBS, transferrin (300 μg/ml),glutamine (2 mM), VEGF (5 ng/ml) and EPO (2 units/ml). The EB cells werecultured in a final volume of 1 ml in a 35 mm bacterial grade dishes ina humidified environment of 5% CO₂ at 37° C. The EB cells were culturedfor about 7 days.

The resulting "mixed" cell population was viewed under an inverted Leitzmicroscope. Under the above culture conditions, 3 differentmorphological types of cell colonies arose and representative cells areshown in FIG. 4. A first cell type, indicated as cell A in FIG. 4,consisted of an erythroid cell having the typical characteristics of adistinct compact cluster of small cells having red color. A second celltype, indicated as cell B in FIG. 4, consisted of a spherical cellhaving a larger size than an erythroid cell, such as cell A. A thirdcell type, indicated as cell C in FIG. 4, consisted of a spherical cellhaving a similar size as an erythroid cell but having a single longprocess extending from the cell.

B. Kinetics of Mixed Erythroid and Endothelial Population Development

Varying EB cell populations were generated using the method describedabove in section A by incubating ES cells for 4, 5, 6 and 7 days.Various types of colonies were then scored about 7 days followingplating based on the 3 different morphologies described in Section A andcultures containing erythroid (ERY) and non-erythroid (NON-ERY) cellswere scored as "mixed" (MIX) populations. Referring to FIG. 13, theresults indicate that the number of mix colonies decreases withincreasing age of EB cells and are almost undetectable by day 7.Meanwhile, erythroid colonies increased in number between days 4 and 6of differentiation.

C. Influence of Specific Growth Factors on Mixed Erythroid andEndothelial Population Development

An EB cell population was generated using the method described above insection A by incubating ES cells for 4 days. In a first experiment, theeffect of specific growth factors on the development of mixedendothelial and erythroid cell populations was tested. Separatepopulations of Day 4 EB cells were plated in the presence of EPO (2units/ml) alone, VEGF (5 ng/ml) alone, a mixture of EPO and VEGF, or nofactor. The cultures were then scored for erythroid (ERY), non-erythroid(NON-ERY) cells and mixed (MIX) cell populations as described above.Referring to FIG. 14, the results indicate that the combination of EPOand VEGF growth factors induced the best EB cell differentiation, inparticular to mixed cell populations.

In a second experiment, the concentration of specific growth factors onthe development of mixed endothelial and erythroid cell populations wastested. Separate populations of Day 4 EB cells were plated in thepresence of 0 nanograms (ng) per ml of EPO+VEGF (E/V/0), 5 ng/mlEPO+VEGF (E/V/5), 10 ng/ml EPO+VEGF (E/V/10), 15 ng/ml EPO+VEGF(E/V/15), 30 ng/ml EPO+VEGF (E/V/30), and no factor (-F). The cultureswere then scored for erythroid (ERY), non-erythroid (NON-ERY) cells andmixed (MIX) cell populations as described above. Referring to FIG. 15,the results indicate that 5 ng/ml of the combination of EPO and VEGFgrowth factors induced the best EB cell differentiation, in particularto mixed cell populations.

D. von Willebrand Factor and Acetylated-LDL Staininq of Mixed CellPopulations

A mixed population of cells derived from the method described in SectionA was stained with the endothelial cell specific marker von Willebrandfactor (vWF) and diI-Acetylated-low density lipoproteins (DiI-Ac-LDL).The mixed colonies were picked from methyl cellulose culture and allowedto adhere overnight to cover slips that were coated with poly-L-lysineor gelatin. Cells were stained for vWF by the following method. Cellsattached to cover slips were fixed for 10 minutes in a solutioncontaining 3% paraformaldehyde and 3% sucrose in PBS. After washing 2 to3 times, the cells were permeabilized with 0.2% Triton X-100 in PBS. Asolution of normal mouse serum and human immunoglobulins was used toblock non-specific binding. Subsequently, the cells were incubated witha rabbit anti-human vWF antibody for 1 hour and washed 5 times, 5minutes each wash. The cover slips were then incubated with horse radishperoxidase labelled goat anti-rabbit antibody (obtained from Fisher) for1 hour and washed for 5 times, 5 minutes each wash. The labelled cellswere then incubated for 15 minutes in a solution containing 0.5 mg/ml ofdiaminobenzidine, 3 mg/ml nickel sulfate, 0.003% H₂ O₂, and 100 mM Tris(pH 7.5).

The vWK stained cells were analyzed by eye. The results indicated thattwo of the three cell types identified in the mixed population(described above in Section A) were labelled with vWF. A representativefiled of stained cells is shown in FIG. 16. Referring to FIG. 16A, thecells described in Section A that had long processes stained with vWF.Referring to FIG. 16B, the cells described in Section A that were largerthan the red erythroid cells also stained with vWF. The rederythroid-like cells identified in section A sis not stain with vWF.Referring to FIG. 16C, a control population of macrophage cells alsofailed to stain with vWF. These results indicate that the non-erythroidcells in the mixed population having long processes or having a largersize than the erythroid cells stain with an endothelial cell specificmarker.

Cells were tested for LDL uptake by the following method. Ten μg/mlDiI-Ac-LDL was added to the medium of cultures of mixed cell populationsand incubated for 4 hours at 37° C. Following incubation, the cells werewashed 3 times and fixed with a solution of 3% paraformaldehyde and 3%sucrose in PBS. Following fixation, the cells were mounted on glassslides using 90% glycerol and 10% PBS and analyzed by eye. The resultsindicated that the non-erythroid cells in the mixed population havinglong processes or having a larger size than the erythroid cells took upthe DiI-Ac-LDL, an endothelial cell specific stain.

Taken together, the observation that the non-erythroid cells generatedin the presence of VEGF stained with both vWF and DiI-Ac-LDL indicatesthat the non-erythroid cells represent endothelial cells and thereforethe mixed colonies appear to contain both hematopoietic and endothelialcells.

Example 11

This example demonstrates that erythroid and endothelial cells in amixed population arose from a common precursor cell.

EB cell colonies were generated using the method described in Example 1.To determine if the differentiation of EB cells in the presence of VEGFand EPO was significantly reduced in GATA-2⁻ EB cells, varying numbersof GATA-2⁺ EB cells (see FIG. 17) were plated into Methyl cellulosecultures containing VEGF, EPO and 2×10⁵ GATA-2⁻ EB cells (as feedercells; kindly provided by Dr. Stuart Orkin at Children's hospital inBoston). The cultures were then analyzed for the development of mixedcolonies under an inverted Leitz microscope. It was found that when astandard number of GATA-2⁻ EB cells were added to cultures containingvarying numbers of GATA-2⁺ EB cells, a linear relationship between thenumber of mixed colonies and the number of GATA-2⁺ EB cells wasobserved. When plotted in a log:log format, the slope of thisrelationship was not significantly different from 1, a finding whichindicates that the colonies derive from a single cell (see FIG. 17).

Example 12

This example describes confirmation studies of the single cell origin ofmixed colonies using a retroviral marking technique.

Retroviruses that carry unique inserts in addition to G418 resistantmarker (retroviral vector LNCX based) were used to mark EB cellpopulations generated according to the method of Example 1. Day 4 EBcell populations were dissociated with trypsin and incubated withretroviruses in the presence of polybene (5 μg/ml), VEGF, EPO, andIGF-1. After 5-8 hours, the cells were incubated in a 1% methylcellulose culture with VEGF, EPO and G418 (150 μg/ml). Cells wereroutinely dispersed using a syringe attached to a 20 gauge needlebecause the cells tended to aggregate during viral infection. Theresulting mixed colonies were picked into 0.1× PBS and lysed for 8minutes at 95° C., treated with proteinase K, heat denatured for 8minutes at 95° C., and subjected to PCR amplification. LNCX retroviralvector sequence was used for the cDNA amplification. An upstream primer(5'-CGCGGCCCCAAGCTTGTTAACATCGATGGATG-3'; SEQ ID NO:3) and a downstreamprimer (5'-GGCGTTACTTAAGCTAGCTTGCCAAAGGTAC-3'; SEQ ID NO:4) were used.PCR products were gel purified and subjected to further amplification.After PCR amplification, excessive primers were removed by filtrationthrough centricon 30 filters and the DNA was concentrated by ethanolprecipitation. The presence of the insert sequence at the junction ofcDNA insert and LNCX retroviral vector was analyzed in 7 G418 resistantmixed colonies by sequencing using standard dideoxy sequencing methods.

The sequence analysis indicated that all of the G418 resistant mixedcolonies contained the same insert sequence, thereby indicating that themixed colonies arose from a single clone.

In summary, the results from experiments involving mixed endothelial anderythroid cell population formation indicate that there is a closeassociation of erythroid and endothelial cell development from a commonprecursor. A mixed erythroid and non-erythroid cell population can begenerated using the growth factors VEGF and EPO. The relationshipbetween the number of mixed colonies generated and the number of cellsplated was linear and individual mixed colonies marked with uniqueretroviruses revealed that these colonies mixed colonies are derivedfrom a common precursor that gave rise to both blood and endothelialcells.

Example 13

This example describes the production of HOX11 immortalized precursorcell populations.

A. Preparation of Recombinant MSCV-HOX11 Retrovirus expression vector.

The plasmid MSCV-HOX11 (described in Hawley et al., Oncogene 9:1-12,1994) was constructed by blunt-end ligating a 1,152 base pair (bp) ApaIfragment (positions 231 to 1382) of the human HOX11 (referred to hereinas TCL-3) cDNA (described in detail in Lu et al., EMBO J. 10:2905-2910,1991) into the HpaI site of the MSCVv2.1 retroviral vector (described inHawley et al., J. Exp. Med. 176:1149-1163, 1992; Hawley et al., GeneTherapy 1:136-138, 1994), which contains a neomycin phosphotransferase(neo) gene conferring resistance to G418 (Geneticin, Life Technologies).

To produce replication-defective recombinant virus, MSCV-HOX11 plasmidwas linearized by digestion with NdeI and then electroporated intoGP+E-86 ecotropic helper-free packaging cells (described in Markowitz etal., J. Virol. 62:1120-1124, 1988) using the methods described in Hawleyet al. (Plasmid 22:120-131, 1989). The electroporated cells were thencultured for about 24 hours. Cell-free (filtered through a 0.45-μmfilter) supernatant was then collected and used to infecttunicamycin-treated (0.1 μg/ml for 16 hours) GP+E-86 cells (described inHawley et al., Leukemia Res. 15:659-673, 1991). The infected cells werecultured in Dulbecco's modified Eagle medium (DMEM) containing about 400μg/ml G418 for about 11 days. Following selection, about 100 colonieswere pooled and propagated as a mass culture (referred to herein asGP+E-86/MSCV-HOX11 cells). GP+E-86/MSCV-HOX11 cells were maintained inDMEM containing 4.5 g/l glucose and 10% calf serum in a humidifiedatmosphere containing 5% CO₂ at 37° C. Control GP+E-86/MSCVv2.1 cellsexporting parental MSCVv2.1 virus were similarly generated.

The GP+E-86/MSCV-HOX11 and GP+E-86/MSCVv2.1 were transferred to DMEMmedium containing 150 μM monothioglycerol and 15% heat inactivated fetalcalf serum and cultured until the cultures were subconfluent (about 24hours). Virus-containing containing supernatants were then collected,filtered through 0.45-μm filters and used immediately to infect CCEembryonic stem (ES) cells. GP+E-86/MSCV-HOX11 and GP+E-86/MSCVv2.1producers have titers of 4-8×10⁶ colony-forming units/ml when assayed onNIH3T3 fibroblasts in the presence of 400 μg/ml G418.

B. Infection of CCE ES Cells with MSCV-HOX11 Plasmid

The method for preparing an EBHX cell population is schematicallyillustrated in FIG. 18.

CCE ES cells were retrieved from frozen stocks of cells that had beencultured for about 17 passages (about 30 days). The thawed cells werecultured for one passage (about 2 days) in DMEM medium containing 150 μMmonothioglycerol and 15% heat inactivated fetal calf serum (referred toherein as growth medium) in the presence of LIF) on 0.1% gelatinizeddishes (described in Keller et al., Mol. Cell. Biol. 13:473-486, 1993).The culture medium was removed from the monolayer of ES cells.Separately, about 1 ml of freshly harvested virus-containingsupernatants from GP+E-86/MSCV-HOX11 cells or 1 ml of freshly harvestedvirus-containing supernatants from GP+E-86/MSCVv2.1 cells, eachsupplemented with 1% LIF conditioned medium and 8 μg/ml polybrene(Sigma, St. Louis, Mo.) was added to about 4×10⁵ cells. The cells wereincubated at 37° C. for 2 hours with occasional rocking (about everyhalf hour). After about 2 hours, 4×volume DMEM growth medium was addedand the incubation continued overnight. The infection was repeated thenext day. The doubly infected population was then expanded and culturedin the presence of 500 μg/ml G418 for about 2 days. The medium was thenchanged and the cells further cultured for about another 24 hours. Thecells were trypsinized and recovered. One half of each culture wasfrozen at -80° C. The remainder of the cells were cultured for about 48hours in G418-supplemented DMEM growth medium and then replated at a 1:8dilution (aliquots were also frozen at -80° C. at this time). Thereplated cells were then cultured for about 7 days in G418-supplementedDMEM growth medium, the cells were harvested (aliquots were also frozenat -80° C. at this time).

Example 14

This example describes the selection of EBHX cell populations of thepresent invention from HOX11 transformed ES cell populations.

EB-HOX11 transformed cell populations were generated using the followingmethod. HOX11 transformed ES cells described in Example 13 weretrypsinized, washed and counted using techniques standard in the art.The freshly dissociated transformed cells were then cultured in IMDMcontaining 15% platelet-derived fetal bovine serum (PDS; obtained fromAntech, Tex.; also referred to herein as platelet-poor fetal bovineserum, PP-FBS), 4.5×10⁻⁴ M MTG, transferrin (300 μg), glutamine (2 mM).The HOX11 transformed cells were plated in a final volume of 10 ml at aconcentration of about 3000 to about 4500 cells per ml of medium in 100mm bacterial grade dishes. The HOX11 transformed cell population wasthen cultured in a humidified environment of 5% CO₂, at a temperature of37° C. for about 7 days to form an EB-HOX11 transformed cell population.Following the 7 day incubation, EB-HOX11 transformed cell populationswere harvested using standard techniques.

Cells from EB-HOX11 transformed cell populations were then furtherdifferentiated into EBHX populations of the present invention using thefollowing method (see FIG. 18). A series of cultures were prepared byplating about 5×10⁵ EB-HOX11 transformed cells and culturing the cellsin liquid culture comprising IMDM containing 10% pre-selected normalFCS, transferrin (300 μg/ml), glutamine (2 mM), and either a mixture ofC-kit ligand (100 ng/ml) and EPO (2 units/ml) or IL-3 (saturatingamounts) and EPO (2 units/ml). The EB-HOX11 transformed cells werecultured in a final volume of 1 ml in a 100 mm bacterial grade dishes ina humidified environment of 5% CO₂ at 37° C. for from about 1 day toabout 100 days.

At specific times, cells were cultured in a first methyl celluloseculture step comprising 1% methyl cellulose containing either a mixtureof C-kit ligand (100 ng/ml) and EPO (2 units/ml) or IL-3 (saturatingamounts) and EPO (2 units/ml). Individual colonies were then recoveredfrom this culture and expanded. The expanded colonies are referred toherein as EBHX cell populations.

Portions of EBHX cell populations from this first methyl cellulose stepwere either frozen at -70° C. and stored, or further cultured in asecond methyl cellulose step comprising 1% methyl cellulose containingeither a mixture of C-kit ligand (100 ng/ml) and EPO (2 units/ml) orIL-3 (saturating amounts) and EPO (2 units/ml). Individual colonies werethen recovered from this culture, expanded and either frozen ormaintained in culture.

Referring to FIG. 18, EBHX-1 through EBHX-7 (also referred to herein asLines 1 through 7) of the present invention were generated from coloniesrecovered from the second methyl cellulose step comprising a mixture ofIL-3 and EPO. EBHX-8 through EBHX-11 (also referred to herein as Lines 8through 11) of the present invention were generated from coloniesrecovered from the second methyl cellulose step comprising a mixture ofC-kit ligand and EPO. EBHX-14 and EBHX-15 (also referred to herein asLines 14 and 15) of the present invention were generated from coloniesrecovered from the first methyl cellulose step. EBHX-14 was generatedfrom the methyl cellulose culture comprising a mixture of IL-3 and EPO,and EBHX-15 was generated from the methyl cellulose culture comprising amixture of C-kit ligand and EPO. Following recovery, EBHX-15 wassubsequently maintained in a culture comprising a mixture of IL-3 andEPO.

Example 15

This example describes the identification of cell surface markers onHOX11 immortalized pluripotent cell populations.

Approximately 2×10⁵ EBHX-1, EBHX-4, EBHX-11, EBHX-14 or EBHX-15 cellswere incubated for 30 min on ice with 0.2 ml of culture supernatantcontaining anti-FcγRII/FcγRIII antibody from the 2.4G2 hybridoma (ATCC)to block FcγRII and FcγRIII on the surface of the cells. The cells werethen washed 3 times and incubated for 30 min on ice separately with oneof the following antibodies directly labeled with FITC or biotinylated:anti-CD45 antibody (1:1000; a gift from John Cambier, National JewishCenter, Denver, Colo.), anti-Aa4.1 antibody (1:25; a gift from JohnMcKearn, Monsanto, St. Louis, Mo.), anti-Sca-1 antibody (1:10; a giftfrom Jan Klein, Max-Planck Institute, Tubingen, Germany), anti-HSAantibody (1:10; a gift from John Cambier), anti-FcγRII/FcγRIII antibody(1:100; obtained from Pharmingen), anti-Thy-1 antibody (1:100; obtainedfrom Pharmingen), anti-B220 antibody (1:100; obtained from Pharmingen),anti-Mac-1 antibody (1:20; obtained from Boehringer Mannheim, Montreal,PQ), anti-Gr-1 antibody (1:100; obtained from Pharmingen), anti-CD44antibody (1:1000; Patrice Hugo, Research Institute of Montreal,Montreal, Canada), anti-VLA-4α antibody (1:10; obtained from ATCC) andanti-LFA-1β antibody (1:1000; obtained from Pharmingen). Cells incubatedwith biotinylated anti-Aa4.1, anti-Sca-1, anti-HSA and anti-VLA-4αantibodies were washed 3 times and counterstained withstreptavidin,R-phycoerythrin conjugate (1:100; Molecular Probes, Inc.,Eugene, OR),

Approximately 2×10⁵ EBHX-1, EBHX-4, EBHX-11, EBHX-14 or EBHX-15 cellswere incubated for 30 min on ice separately with either anti-VLA-4α,anti-ICAM-1 antibody (1:2 culture supernatant; obtained from ATCC) oranti-TER119 antibody (1:40; obtained from Pharmingen). The cells werethen washed 3 times and labeled with FITC-conjugated goat F(ab')₂anti-rat IgG (1:100; obtained from Jackson ImmunoResearch, West Grove,Pa.) for 30 min on ice, and analyzed on a Epics Elite flow cytometer(Coulter Electronics, Hialeah, Fla.).

The mean linear fluorescence signals obtained for cells incubated withall antibodies is shown in Table 2.

                                      TABLE 2    __________________________________________________________________________    Surface Marker Analysis of HOX11-Expressing Cell Populations    Marker     EBHX-1                     EBHX-4                           EBHX-11                                 EBHX-14                                       EBHX-15    __________________________________________________________________________    CD45 (Ly-5)                0.9 (1.61)                      0.4 (1.99)                            0.2 (0.61)                                  3.1 (2.26)                                       84.8 (9.38)    Aa4.1*      0.2 (0.79)                      0.3 (0.79)                            0.1 (0.65)                                  7.2 (1.13)                                       18.6 (3.25)    Sca-1 (Ly6A/E)*                0.4 (0.94)                      0.4 (0.80)                            0.1 (0.62)                                  5.0 (1.38)                                        1.1 (2.13)    HSA (CD24)*                0.1 (0.93)                      0.2 (0.79)                           99.8 (6.55)                                  4.6 (2.16)                                       98.5 (12.5)    FcγRII/III (CD32/CD16)               78.3 (3.66)                     85.6 (5.44)                            0.3 (0.55)                                 91.1 (5.41)                                       99.7 (39.8)    Thy-1       6.6 (4.81)                      3.2 (4.24)                            0.3 (0.53)                                 86.3 (30.1)                                       95.7 (63.3)    B220 (CD45R)                0.7 (2.48)                      0.6 (2.14)                            0.3 (0.54)                                  0.5 (1.60)                                        0.4 (7.82)    Mac-1 (CD11b)                0.7 (1.68)                      0.2 (1.45)                            0.3 (0.56)                                  4.0 (2.65)                                       11.8 (12.1)    Gr-1 (Ly-6G)                0.7 (1.59)                      0.5 (2.13)                            0.3 (0.48)                                 21.6 (19.7)                                        6.3 (31.5)    CD44       91.6 (8.82)                     94.5 (10.2)                           98.8 (9.08)                                 92.4 (7.48)                                        9.9 (26.6)    VLA-Aα (CD49d)*               72.4 (1.45)                     98.0 (4.00)                           85.8 (1.06)                                 99.7 (5.35)                                       99.5 (6.27)    LFA-1β (CD18)               96.9 (6.03)                     98.4 (7.17)                            0.3 (0.52)                                 98.7 (9.72)                                       98.5 (23.7)    ICAM-1 (CD54)†                1.3 (1.82)                      0.5 (1.64)                           27.8 (0.78)                                  0.5 (1.44)                                       27.7 (9.2)    TER119†                6.2 (1.80)                      0.9 (1.73)                            0.3 (0.73)                                 12.1 (4.15)                                       24.8 (8.59)    __________________________________________________________________________     Results are the percentages of labeled cells (log mean fluorescence     intensity) determined by direct staining of FcγRII/IIIblocked cells     with FITCconjugated antibodies     *direct staining of FcγRII/IIIblocked cells with biotinylated     antibodies and PEstreptavidin

The results indicate that EBHX-1 cells express FcγRII and/or FcγRIII,Thy-1, CD44, VLA-4α and LFA-1β on their surface. The results usinganti-TER119 antibody indicated that TER119 protein was most likelyexpressed on the surface of EBHX-1 cells. The results also indicatedthat EBHX-4 cells express FcγRII and/or FcγRIII, CD44, VLA-4α and LFA-1,β on their surface. The results using anti-Thy-1 antibody indicated thatThy-1 protein was most likely expressed on the surface of EBHX-4 cells.Results obtained for EBHX-11 cells indicated that such cells expressHSA, CD44, VLA-4α, LFA-1β and ICAM-1 on their surface. Moreover, resultsobtained for EBHX-14 cells indicated that such cells express CD45,Aa4.1, Sca-1, HSA, FcγRII and/or FcγRIII, Thy-1, Mac-1, Gr-1, CD44,VLA-4α and LFA-1β on their surface. The results using anti-TER119antibody indicated that TER119 protein was most likely expressed on thesurface of EBHX-14 cells. Results obtained for EBHX-15 cells indicatedthat such cells express CD45, Aa4.1, HSA, FcγRII, FcγRIII, Thy-1, Mac-1,Gr-1, CD44, VLA-4α, LFA-1β, ICAM-1 on their surface.

Example 16

This example describes the growth factor responsiveness of HOX11immortalized pluripotent cell populations.

Approximately about 1×10⁵ to about 10×10⁵ EBHX-1, EBHX-11 or EBHX-14cells were cultured in IMDM containing 15% PP-FBS, transferrin (300μg/ml), glutamine (2 mM) and either IL-3 (saturating amounts) EPO (2units/ml), C-kit ligand (100 ng/ml), LIF (10 ng/ml), IL-4 (saturatingamounts), IL-6 (5 ng/ml), IL-11 (25 ng/ml), VEGF (5 ng/ml), GM-CSF 20U/ml), M-CSF (100 U/ml), G-CSF (1000 U/ml) or in the absence of anyfactor. The EBHX cells were cultured in a final volume of 1 ml in a 35mm bacterial grade dishes in a humidified environment of 5% CO₂ at 37°C. The EBHX cells were cultured for about 7 to about 10 days.

Factor responsiveness to each EBHX cell population tested was determinedby counting the number of colonies formed per about 1×10⁵ to about10×10⁵ cells plated. The colony counts in the presence of a factor wasthen compared with the colony counts obtained for cells cultured in theabsence of any factor. The results obtained for EBHX-1 cells areillustrated in FIG. 19 and indicate that EBHX-1 cells (Line #1) respondto a combination of IL-3 and EPO, EPO alone, IL-3 alone, c-kit ligand,LIF, IL-4, IL-6 and IL-11. In addition, the results obtained for EBHX-11cells are illustrated in FIG. 20 and indicate that EBHX-11 cells (Line#11) respond to a combination of c-kit ligand and EPO, EPO alone, c-kitligand alone, IL-3 and LIF. Moreover, the results obtained for EBHX-14cells are illustrated in FIG. 21 and indicate that EBHX-14 cells (HOXLine 14) respond to a combination of IL-3 and EPO, IL-3 alone andGM-CSF.

Example 17

This example describes the expression of βH1, ζ and β major globin genesby EBHX-1 and EBHX-11 cells.

cDNA was prepared from mRNA isolated from approximately EBHX-1 orEBHX-11 cells using an oligo dT primer under standard conditions. ThecDNA was resolved by gel electrophoresis and immobilized on a nylonmembrane using methods standard in the art and described in Sambrook etal. (ibid.). The membranes were then hybridized in the presence of a ³²P-labelled βH1-globin oligonucleotide probe (about 5×10⁶ cpm/ml ofhybridization buffer; disclosed in Hill et al., J. Biol. Chem.259:3739-3744, 1984), a ³² P-labelled β major-globin oligonucleotideprobe (about 5×10⁶ cpm/ml of hybridization buffer; disclosed in Konkelet al., Cell 15:1125-1132, 1978) or a ³² P-labelled ζ-globinoligonucleotide probe (about 5×10⁶ cpm/ml of hybridization buffer;disclosed in Leder et al., Mol. Cell. Biol. 5:1025-1033, 1985), usingChurches hybridization buffer (containing 1 mM EDTA, 0.5M Na₂ HPO₄ at pH7.2 and 7% SDS) for 18 hours at 42° C. The blots were then washed twicein wash buffer (containing 1 mM EDTA, 40 Na₂ HPO₄ at pH 7.2 and 1% SDS)for 5 min at room temperature, and twice for 10 min at 42° C. Theresulting blot was exposed to X-ray film to produce an autoradiogram.

The autoradiogram illustrated that βH1, ζ and β major-globin RNA wasexpressed in both EBHX-1 and EBHX-11 cells.

Taken together, the results described in Examples 13-17 indicate thatthe HOX11 immortalized cells of the present invention represent cellsearly in the hematopoietic lineage.

Example 18

This example describes the production of endothelial cell conditionedmedium.

A. Production of an endothelial cell population from an EB cellpopulation.

ES cells were prepared and cultured under conditions described inExample 1 for about 4 days to form a 4 day EB cell population. The EBcell population was then infected with retrovirus comprising DNAencoding Polyoma Middle T antigen and a neo gene conferring resistanceto G418 using standard conditions (described in detail in Williams etal., ibid.). The retrovirus transformed EB cells were then cultured forabout 7 to about 10 days in IMDM medium containing about 500 μg of G418and about 5% to about 10% normal FCS. Surviving cells were then replatedin culture medium lacking G418 and containing endothelial cell growthsupplement (about 50 to 100 μg/ml of culture medium; CollaborativeResearch, Bedford, Mass.) and cultured under standard conditions forabout 2 months. Rapidly growing cells from this population, referred toherein as D4T, were recovered and frozen at -70° C. and stored.

D4T cells were then stained for FACS analysis using either anti-Flk-1antibody (obtained from Warner Risan, Bad Nauheim, Germany) or anti-CD31antibody (obtained from Pharmingen). The results from the FACS analysisindicated that D4T cells express both CD31 and Flk-1, thus indicatingthat the D4T cells possessed endothelial cell characteristics.

D4T cells were also tested for DiI-Ac-LDL uptake using methods describedin Example 10. The results indicate that the D4T cells stained withDiI-Ac-LDL, confirming the results of the FACS analysis and indicatingthat the D4T cells posses endothelial cell characteristics.

B. Production of Conditioned Medium

About 5×10⁴ D4T cells were cultured in IMDM medium comprising about 10%normal FCS and about 50 to 100 μg/ml of ECGF for about 72 hours at 37°C. The supernatant from the culture was recovered and filtered through a0.2 micron filter.

C. Stimulation of BLAST Cell Population Growth Using Conditioned Medium.

Two separate series of 3 day EB cell (prepared according to the methodin Example 1) cultures were prepared at cell densities of about 6×10³,about 1×10⁴, about 1.5×10⁴, about 3×10⁴ or about 5×10⁴ cells perculture. The cultures were prepared and grown under conditions for BLASTcell colony formation (described in Example 3) using IMDM mediumcontaining about 10% pre-selected FCS, EPO (2 units/ml), C-kit ligand(100 ng/ml), VEGF (5 ng/ml), and either in the presence or absence of25% final volume of D4T conditioned medium. The EB cells were culturedfor about 4 days under standard conditions and the number of BLAST cellcolonies were determined.

The results (shown in FIG. 22) indicate that the addition of conditionedmedium (+CM) in the culture medium improved BLAST cell growth comparedwith growth in the absence of conditioned medium (-CM), in particular atthe lower cell densities. Thus, the D4T conditioned medium contains oneor more compounds capable of enhancing the growth of BLAST cells from EBcells.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TGGTGGAGTCTGGGGGAGGCTTA23    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GGCTCCCTCAGGGACAAATATCCA24    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CGCGGCCCCAAGCTTGTTAACATCGATGGATG32    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GGCGTTACTTAAGCTAGCTTGCCAAAGGTAC31    __________________________________________________________________________

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

What is claimed:
 1. A pluripotent cell population wherein said cellpopulation is transformed with a HOX11 gene, and wherein said cellpopulation differentiates into cellular lineages including primitiveerythroid cells and definitive erythroid cells.
 2. The cell populationof claim 1, wherein said cell population expresses a cell surfacemolecule selected from the group consisting of FcγRII, FcγRIII, Thy-1,CD44, VLA-4α, LFA-1β and combinations thereof, and is responsive to agrowth factor selected from the group consisting of interleukin-3,interleukin-4, interleukin-6, interleukin-11, erythropoietin, C-kitligand, leukocyte inhibitory factor and mixtures thereof.
 3. The cellpopulation of claim 1, wherein said cell population expresses a cellsurface molecule selected from the group consisting of HSA, CD44,VLA-4α, LFA-1β, ICAM-1 and combinations thereof, and is responsive to agrowth factor selected from the group consisting of interleukin-3,erythropoietin, C-kit ligand, leukocyte inhibitory factor and mixturesthereof.
 4. The cell population of claim 1, wherein said cell populationexpress βH1, ζ and β major-globin RNA.
 5. The cell population of claim1, wherein said cell population comprises precursor cells whichdifferentiate into erythroid lineages, and leukocyte lineages.
 6. Thecell population of claim 1, wherein said cell population is derived froman embryonic stem cell population.
 7. The cell population of claim 1,wherein said cell population is derived from a transformed embryoid bodycell population, said transformed embryoid body cell population beingderived by culturing an embryonic stem cell population transformed witha HOX11 gene in an embryonic body cell medium.
 8. The cell population ofclaim 7, wherein said embryoid body cell medium comprises about 15%serum selected from the group consisting of platelet-poor fetal bovineserum and pre-selected normal fetal calf serum.
 9. The cell populationof claim 7, wherein said embryoid body cell medium does not includeleukocyte inhibitory factor.
 10. The cell population of claim 7, whereinsaid step of culturing is performed from about 1 day to about 28 days.11. The cell population of claim 7, wherein said step of culturing isperformed from about 3 days to about 8 days.
 12. The cell population ofclaim 1, wherein said cell population is derived from an embryoid bodycell population transformed with a HOX11 gene cultured under effectiveconditions in an effective medium comprising serum selected from thegroup consisting of platelet-poor fetal bovine serum and pre-selectednormal fetal calf serum, and a growth factor selected from the groupconsisting of C-kit ligand, interleukin 3, erythropoietin, andcombinations thereof.
 13. The cell population of claim 1, wherein saidcell population is derived from an embryoid body cell populationtransformed with a HOX11 gene cultured under effective conditions in aneffective medium comprising serum selected from the group consisting ofplatelet-poor fetal bovine serum and pre-selected normal fetal calfserum, and a combination of growth factors selected from the groupconsisting of C-kit ligand combined with erythropoietin, and interleukin3 combined with erythropoietin.
 14. The cell population of claim 1,wherein said cell population is produced by the method comprising:a)introducing a HOX11 gene into an embryonic stem cell population tocreate a modified stem cell population; b) culturing said modified stemcell population for about 7 days in an embryoid body cell medium undereffective conditions to produce a transformed embryoid body cellpopulation; and c) incubating said transformed embryoid body cellpopulation in the presence of a combination of growth factors selectedfrom the group consisting of C-kit ligand combined with erythropoietin,and interleukin 3 combined with erythropoietin.
 15. A HOX11 pluripotentprecursor cell population, wherein said cell population is transformedwith a HOX11 gene, and wherein said cell population differentiates intocellular lineages including primitive erythroid cells and definitiveerythroid cells, and is responsive to a growth factor selected from thegroup consisting of interleukin-3, interleukin-4, interleukin-6,interleukin-11, erythropoietin, C-kit ligand, leukocyte inhibitoryfactor and mixtures thereof.
 16. The cell population of claim 15,wherein said cell population expresses a cell surface molecule selectedfrom the group consisting of FcγRII, FcγRIII, Thy-1, CD44, VLA-4α,LFA-1β and combinations thereof.
 17. The cell population of claim 15,wherein said cell population expresses βH1 globin RNA, ζ globin RNA andβ major-globin RNA.
 18. A HOX11 pluripotent precursor cell population,wherein said cell population is transformed with a HOX11 gene, andwherein said cell population differentiates into cellular lineagesincluding primitive erythroid cells and definitive erythroid cells, andis responsive to a growth factor selected from the group consisting ofinterleukin-3, erythropoietin, C-kit ligand, leukocyte inhibitory factorand mixtures thereof.
 19. The cell population of claim 18, wherein saidcell population expresses a cell surface molecule selected from thegroup consisting of HSA, CD44, VLA-4α, LFA-1β, ICAM-1 and combinationsthereof.
 20. The cell population of claim 18, wherein said cellpopulation expresses βH1 globin RNA, ζ globin RNA and β major-globinRNA.
 21. The cell population of claim 1, wherein said cell populationexpresses a combination of cell surface molecules, said combinationselected from the group consisting of (a) FcγRII, FcγRIII, Thy-1, CD44,VLA-4α, and LFA-1β; (b) FcγRII, FcγRIII, CD44, VLA-4α, and LFA-1β; and(c) HSA, CD44, VLA-4α, LFA-1β, and ICAM-1.
 22. A HOX11 precursor cellpopulation which is transformed with a HOX11 gene and which expressesβH1, ζ and β major-globin RNA.
 23. A HOX11 pluripotent precursor cellpopulation, wherein said cell population is produced by the methodcomprising:a) introducing a HOX11 gene into an embryonic stem cellpopulation to create a modified stem cell population; b) culturing saidmodified stem cell population from about 1 day to about 7 days in anembryoid body cell medium under effective conditions to produce atransformed embryoid body cell population; and c) incubating saidtransformed embryoid body cell population in the presence of acombination of growth factors selected from the group consisting ofC-kit ligand combined with erythropoietin, and interleukin 3 combinedwith erythropoietin to obtain said pluripotent precursor cell ispopulation.